Immunogenic peptides for the treatment of prostate and breast cancer

ABSTRACT

Immunogenic T-cell receptor gamma Alternate Reading Frame Protein (TARP) polypeptides are disclosed herein. These immunogenic TARP polypeptides include nine consecutive amino acids of the amino acid sequence set forth as SEQ ID NO: 9 and do not comprise amino acids 1-26 or amino acids 38-58 of SEQ ID NO: 1. Several specific, non-limiting examples of these polypeptides are set forth as SEQ ID NOs: 3-7. Nucleic acids encoding these polypeptides, and host cells transfected with these nucleic acids, are also disclosed. Methods of using these polypeptides, and polynucleotides encoding these polypeptides, for the treatment of breast and prostate cancer are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a § 371 U.S. national stage of PCT/US2004/017574, filed Jun. 2,2004, which was published in English under PCT Article 2(2), and claimsthe benefit of U.S. Provisional Application No. 60/476,467, filed Jun.5, 2003, which is incorporated by reference herein in its entirety.

FIELD

This application relates to the field of cancer therapeutics,specifically to immunogenic peptides and their use in the treatment ofprostate and breast cancer.

BACKGROUND

Cancer of the prostate is the most commonly diagnosed cancer in men andis the second most common cause of cancer death (Carter and Coffey,Prostate 16:39-48, 1990; Armbruster et al., Clinical Chemistry 39:181,1993). If detected at an early stage, prostate cancer is potentiallycurable. However, a majority of cases are diagnosed at later stages whenmetastasis of the primary tumor has already occurred (Wang et al., Meth.Cancer Res. 19:179, 1982). Even early diagnosis is problematic becausenot all individuals who test positive in these screens develop cancer.

Present treatment for prostate cancer includes radical prostatectomy,radiation therapy, or hormonal therapy. With surgical intervention,complete eradication of the tumor is not always achieved and theobserved re-occurrence of the cancer (12-68%) is dependent upon theinitial clinical tumor stage (Zietman et al., Cancer 71:959, 1993).Thus, alternative methods of treatment including prophylaxis orprevention are desirable.

Breast cancer is the most common type of epithelial cancer among womenin the United States. More than 180,000 women are diagnosed with breastcancer each year. About one in eight women in the United States(approximately 12.8 percent) will develop breast cancer during herlifetime. At present there are no curative therapies available forbreast cancer that has metastasized from its site of origination.

Up to 30% of 180,000 United States patients with potentially curableearly-stage breast and prostate cancer will fail standard surgical orradiotherapy in 2004. In addition, patients with metastatic prostatecancer and the majority of patients with metastatic breast cancer enjoylimited benefit of standard chemotherapy and hormone-based therapies.Immunotherapy may have great potential to improve on these results,combining the tumor specificity of cell-mediated immunity with freedomfrom toxic chemotherapies.

Immunotherapy involves evoking an immune response against cancer cellsbased on their production of target antigens. Immunotherapy based oncell-mediated immune responses involves generating a cell-mediatedresponse to cells that produce particular antigenic determinants, whileimmunotherapy based on humoral immune responses involves generatingspecific antibodies to cells that produce particular antigenicdeterminants.

Recent studies show that immunotherapy of cancer patients may bedramatically improved by the finding that CD8⁺ CTL recognize and killtumor cells that display peptides from tumor-associated antigens withinMHC Class I molecules. In clinical studies it has been found thateffector CD8⁺ T cells play a major role in tumor regression. Severaltumor antigens in prostate cancer models have been identified and HLAallele-specific peptides from those prostate cancer-associated antigenshave been identified as CD8⁺ T cell epitopes. For example, HLA-A2.1binding peptides were described that were derived from prostate specificantigen (PSA) (Correale et al., J Immunol 161:3186, 1998),prostate-specific membrane antigen (PSMA) (Tjoa et al., Prostate 28:65,1996), prostate stem cell antigen (PSCA) (Kiessling et al., Int J Cancer102:390, 2002), and prostate acid phosphatase (Peshwa et al., Prostate36:129, 1998). For PSA, clinical trials are in progress using differentvaccine strategies. However, there clearly is a need to identifyadditional antigens to aid in the diagnosis of prostate cancer, and foruse as additional therapeutic agents.

SUMMARY

Immunogenic T cell receptor gamma Alternate Reading Frame Protein (TARP)polypeptides are disclosed herein. These immunogenic TARP polypeptidesinclude nine consecutive amino acids of the amino acid sequence setforth as SEQ ID NO: 9 and do not comprise consecutive amino acids 1-26or consecutive amino acids 38-58 of SEQ ID NO: 1. Several specific,non-limiting examples of these polypeptides are set forth as SEQ ID NOs:3-7. Nucleic acids encoding these polypeptides, and host cellstransfected with these nucleic acids, are also disclosed.

The polypeptides are of use in generating an immune response to TARP,such as, but not limited to, a T cell response. Specifically, thesepolypeptides can be used to generate an immune response to breast cancerand prostate cancer cells that express TARP.

In one embodiment, the immunogenic TARP polypeptides, or a nucleic acidencoding the polypeptide, are administered to a subject to produce animmune response to TARP. In another embodiment, specific antigenpresenting cells are prepared by contacting dendritic cells with animmunogenic TARP polypeptide. These antigen presenting cells areadministered to a subject. In a further embodiment, the antigenpresenting cells are used to generate cytotoxic T cells thatspecifically recognize TARP. The cytotoxic T cells are administered to asubject. For each of these embodiments, the subject can have breastcancer or prostate cancer, and the cells of the cancer express TARP.

Also disclosed herein is a reagent that includes a tetramer of theimmunogenic TARP polypeptide bound to HLA-A.2.1, wherein the reagent islabeled or unlabeled. This reagent is of use in identifying CD8+ cellsthat specifically bind TARP.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C are polypeptide sequences of TARP (SEQ ID NO: 1), wild-typeHLA-A2 epitopes (amino acids 2-9, 22-30, 29-37 and 27-25 of SEQ ID NO:1), and a line graph showing their binding affinities to HLA-A2.1molecules. FIG. 1A is the amino acid sequence of the TARP protein (SEQID NO: 1). FIG. 1B is the sequence of the predicted-HLA-A2.1-bindingpeptides (amino acids 2-9, 22-30, 29-37 and 27-25 of SEQ ID NO: 1). FIG.1C is a line graph of a T2 binding assay. Peptides were dissolved indouble-distilled water (DDW) or 20% DMSO in DDW, and then differentconcentrations of peptides were added into the culture of TAP-deficientT2 cells. After overnight culture of the cells in medium supplementedwith β-2 microglobulin, cells were stained with anti-HLA-A2. Each assaywas performed in triplicate assay and data in this figure arerepresentative of two experiments with similar results.

FIGS. 2A-C are line graphs and data plots from fluorescence activatedcell sorting experiments. Immunization with peptide-loaded dendriticcell (DC) or a DNA plasmid expressing TARP results in peptide-specificCD8⁺ T cell responses in A2K^(b) transgenic mice. To obtain the data forthe graph shown in FIG. 2A, mice were immunized subcutaneously withpeptide-loaded (10 μM of TARP-29-37 or TARP-27-35) bone marrow-derivedDC and boosted twice at three-week intervals. To obtain the data for thegraph shown in FIG. 2B, mice were immunized with 100 μg of DNA plasmidintramuscularly and boosted four times at three-week intervals. For thedata shown in FIGS. 2A and 2B, after the final immunization, spleen CD8⁺T cells were restimulated with splenocytes in media containing 10 μMsoluble peptides for 7 days. In a 5 hour ⁵¹Cr-release assay, Jurkatcells transfected with HLA-A2 were labeled with ⁵¹Cr, and then pulsedwith 10 μM of peptides. Without washing, target cells were mixed withdifferent numbers of effector cells for 5 hours before harvesting. Forthe data plots shown in FIG. 2C, for intracellular cytokine staining,CD8⁺ T cells from the mice immunized with peptide-pulsed DC werestimulated with the identical peptide and treated with Brefeldin A for 5hour, and then cells were stained with anti-CD8-FITC and IFN-γ-PE asdescribed in the manufacturer's protocol (Pharmingen). In eachexperiment, pooled-spleen CD8⁺ T cells of 3 mice were tested. Data arerepresentative of two repeated experiments with similar results(SE<10%).

FIGS. 3A-C are peptide sequences and two line graphs showing HLA-A2.1epitope-enhancement by amino acid substitutions in the wild-typepeptides. FIG. 3A is the amino acid sequences of predicted enhancedepitopes (SEQ ID NO: 4, wherein L is at position 2, A is at position 3or wherein V is at position 9, SEQ ID NO: 5 and SEQ ID NO: 6). FIGS. 3Band 3C are graphs showing the binding affinity of the substitutedTARP27-35 peptides (FIG. 3B) or the substituted TARP27-37 peptides (FIG.3C) to HLA-A2 molecules. Peptides were dissolved in double-distilledwater or 20% DMSO, and then different concentrations of peptides wereadded into the culture of TAP-deficient T2 cells. After overnightculture of the cells in medium supplemented with β2-microglobulin, cellswere stained with anti-HLA-A2. Each assay was performed in triplicate,and data in this figure are representative of two experiments withsimilar results.

FIGS. 4A-D are FACS plots, a bar graph and two line graphs showing theimmunogenicity of the enhanced epitopes in A2K^(b) transgenic mice.A2K^(b) transgenic mice were immunized subcutaneously with the mixtureof peptide and cytokine in adjuvant as described in the Examplessection. For the data plotted in FIG. 4A, two weeks after second boost,pooled-spleen CD8⁺ T cells from 3-4 mice were restimulated with 1.0 μMof each peptide. During restimulation, CD8⁺ T cells were treated withBrefeldin A for 5 hours. Cells were stained with anti-CD8-FITC andIFN-γ-PE as described in the manufacturer's protocol (Pharmingen). FIG.4B is a bar graph showing the cross-reactivity on each peptide(E:T=100:1). Two weeks after the second boost, pooled spleen CD8⁺ Tcells from three to four mice were restimulated with irradiatedsplenocytes in medium containing 1.0 μM each peptide for 7 days. In a5-hour ⁵¹Cr release assay, Jurkat cells transfected with HLA-A2 werelabeled with ⁵¹Cr and then pulsed with peptides. Without washing, targetcells were mixed with different numbers of effector cells and thencultured for 5 hours before harvesting. FIG. 4C is a line graph showingdata obtained when CTLs raised against the individual peptides indicatedwere tested on target cells pulsed with either wild-type TARP29-37(closed symbols) or no peptide (open symbols). Lytic activity is shownas a function of E:T ratio. Bars represent SE in triplicate assay, andbars smaller than the symbols are not shown. FIG. 4D is a line graphshowing the relationship between peptide binding affinity andimmunogenicity of each peptide. Peptide concentrations for fluorescenceindex value=0.5 (FI₅₀) from FIG. 3 were plotted versus the numbers ofIFN-γ-producing cells (see FIG. 4A). Data were reproducible in tworepeated experiments with similar results.

FIGS. 5A-B are plots and a line graph showing that human CD8⁺ CTL raisedby in vitro stimulation kill peptide-loaded target cells. CD8⁺ T cellsfrom a prostate cancer patient were restimulated with 10 μMpeptide-pulsed autologous dendritic cells (DCs). DCs were derived fromthe culture of autologous monocytes in granulocyte macrophagecolony-stimulating factor and interleukin 4. To mature the DCs, CD40ligand (2 μg/ml) was added on day 4, and then cells were furthercultured for 2-3 days before loading peptides. After several cycles ofin vitro restimulation, CD8⁺ T cells were used as effector cells. Forthe results show in FIG. 5A, cells were stained with anti-CD8α orisotype control antibody. For the results shown in FIG. 5B, in 5-hour⁵¹Cr release assay, C1R-A2.1 cells were labeled with ⁵¹Cr and thenpulsed with peptides. Closed and open circles represent target cellspulsed with and without peptides, respectively. Data are representativeof two repeated experiments with similar results.

FIGS. 6A-B are two sets of line graphs showing cross-reactivity of humanCD8⁺ CTL to different HLA-A2 peptide epitopes. Human CD8⁺ CTL wereraised as described in the description above for FIG. 5. For the resultsshown in FIG. 6A, in a 5-hour ⁵¹CR release assay, C1R-A2.01 cells werelabeled with ⁵¹Cr and then pulsed with 10 μM peptides. For the resultsshown in FIG. 6B, specificity and avidity of CD8⁺ T cells specific forTARP29-37-9V to other peptides were tested by using target cells pulsedwith different concentrations of the indicated peptides. Data arerepresentative of two repeated experiments with similar results.

FIGS. 7A-B are a bar graph and plots showing human CD8⁺ CTL raised by invitro restimulation kill human cancer cell line. For FIG. 7A, in a5-hour ⁵¹Cr release assay, LNCaP (prostate cancer cell line;HLA-A2⁺TARP⁺), MCF-7 (breast cancer cell line; HLA-A2⁺TARP⁺), DU145(prostate cancer cell line; HLA-A2⁻TARP⁻), and PC3-TARP (prostate cancercell line transfected with TARP; HLA-A2⁻TARP⁺) were used as targetcells. Target cells were pre-incubated with 1000 ng/ml human IFN-γ for72 hours. Data are representative of two repeated experiments withsimilar results, and error bars (SEM) were calculated from a triplicateassay in one experiment. For the results shown in FIG. 7B, tumor celllines were stained with anti-HLA-A2 after incubation with or withoutIFN-γ for 72 hours.

FIG. 8 is a diagram including a set of plots from two experimentsshowing HLA-A2-tetramers composed of wild-type and enhanced-epitopesrecognize peptide-specific CD8⁺ T cells in human blood samples.Tetramers of HLA-A2 and peptide were made as described in the Examplesection. Peripheral blood mononuclear cells from breast and prostatecancer patients and normal donors were stained with anti-CD8-FITC andtetramer-PE and then analyzed by flow cytometry. A normal donor wastested simultaneously with each patient as a negative control. Data arerepresentative of two repeated experiments with similar results.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is an amino acid sequence of a full-length TCRγ AlternateReading Frame Protein (TARP) protein.

SEQ ID NO: 2 is a nucleic acid sequence encoding a full-length TARPprotein.

SEQ ID NO: 3 is the amino acid sequence of TARP-29-37, which correspondsto amino acids 29-37 of SEQ ID NO: 1.

SEQ ID NO: 4 is the amino acid sequence of TARP-27-35, which correspondsto amino acids 27-35 of SEQ ID NO: 1.

SEQ ID NO: 5 is the amino acid sequence of TARP-29-37-3A, whichcorresponds to amino acids 29-37 of SEQ ID NO: 1, with a singlesubstitution.

SEQ ID NO: 6 is the amino acid sequence of TARP-29-37-9V, whichcorresponds to amino acids 29-37 of SEQ ID NO: 1, with a singlesubstitution.

SEQ ID NO: 7 is the amino acid sequence of TARP-2-9, which correspondsto amino acids 2-9 of SEQ ID NO: 1.

SEQ ID NO: 8 is the amino acid sequence of TARP-22-30, which correspondsto amino acids 22-30 of SEQ ID NO: 1.

SEQ ID NO: 9 is the consensus sequence for an immunogenic TARPpolypeptide. This sequence corresponds to amino acids 27-37 of SEQ IDNO: 1, with zero, one or two substitutions.

DETAILED DESCRIPTION

I. Abbreviations

APC: antigen presenting cell

CTL: cytotoxic T lymphocyte

DC: dendritic cell

DDW: double distilled water

DMSO: dimethyl sulfoxide

E/T: effector to target

FACS: fluorescence activated cell sorting

FITC: fluorescein isothiocyanate

Flt-3L: flt-3 ligand

GM-CSF: granulocyte/macrophage colony stimulating factor

HLA: human major histocompatibility complex

IL-4: interleukin-4

MHC: Major Histocompatibility Complex

PBL: peripheral blook lymphocytes

PBMC: peripheral blood mononuclear cells

PE: phycoerythrin

TARP: TCRγ Alternate Reading Frame Protein

TCR: T cell receptor

TIL: tumor infiltrating lymphocytes

μM: micromolar

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Adjuvant: A vehicle used to enhance antigenicity; such as a suspensionof minerals (alum, aluminum hydroxide, or phosphate) on which antigen isadsorbed; or water-in-oil emulsion in which antigen solution isemulsified in mineral oil (Freund incomplete adjuvant), sometimes withthe inclusion of killed mycobacteria (Freund's complete adjuvant) tofurther enhance antigenicity (inhibits degradation of antigen and/orcauses influx of macrophages). Immunstimulatory oligonucleotides (suchas those including a CpG motif) can also be used as adjuvants (forexample see U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371;6,239,116; 6,339,068; 6,406,705; and 6,429,199).

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. In one embodiment, T cells respond to the epitope, when theepitope is presented in conjunction with an MHC molecule. Epitopes canbe formed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5, about 9, or about 8-10 aminoacids in a unique spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance.

An antigen can be a tissue-specific antigen, or a disease-specificantigen. These terms are not exclusive, as a tissue-specific antigen canalso be a disease specific antigen. A tissue-specific antigen isexpressed in a limited number of tissues, such as a single tissue.Specific, non-limiting examples of a tissue specific antigen are aprostate specific antigen and/or a breast specific antigen. A tissuespecific antigen may be expressed by more than one tissue, such as, butnot limited to, an antigen that is expressed in both prostate and breasttissue. A disease-specific antigen is expressed coincidentally with adisease process. Specific non-limiting examples of a disease-specificantigen are an antigen whose expression correlates with, or ispredictive of, tumor formation, such as prostate cancer and/or breastcancer. A disease-specific antigen can be an antigen recognized by Tcells or B cells.

Amplification: Of a nucleic acid molecule (e.g., a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a specimen. An example of amplification is thepolymerase chain reaction, in which a biological sample collected from asubject is contacted with a pair of oligonucleotide primers, underconditions that allow for the hybridization of the primers to a nucleicacid template in the sample. The primers are extended under suitableconditions, dissociated from the template, and then re-annealed,extended, and dissociated to amplify the number of copies of the nucleicacid. The product of amplification can be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen.

A naturally occurring antibody (e.g., IgG, IgM, IgD) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. However, it has been shown that theantigen-binding function of an antibody can be performed by fragments ofa naturally occurring antibody. Thus, these antigen-binding fragmentsare also intended to be designated by the term “antibody.” Specific,non-limiting examples of binding fragments encompassed within the termantibody include (i) a Fab fragment consisting of the V_(L), V_(H),C_(L) and C_(H1) domains; (ii) an F_(d) fragment consisting of the V_(H)and C_(H1) domains; (iii) an Fv fragment consisting of the VL and VHdomains of a single arm of an antibody, (iv) a dAb fragment (Ward etal., Nature 341:544-546, 1989) which consists of a V_(H) domain; (v) anisolated complimentarity determining region (CDR); and (vi) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region.

Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture (e.g., see U.S. Pat. Nos.4,745,055; 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125,023;Faoulkner et al., Nature 298:286, 1982; Morrison, J. Immunol. 123:793,1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Breast cancer: A neoplastic condition of breast tissue that can bebenign or malignant. The most common type of breast cancer is ductalcarcinoma. Ductal carcinoma in situ is a non-invasive neoplasticcondition of the ducts. Lobular carcinoma is not an invasive disease butis an indicator that a carcinoma may develop. Infiltrating (malignant)carcinoma of the breast can be divided into stages (I, IIA, IIB, IIIA,IIIB, and IV).

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease an activityor antigenicity of an antigenic epitope of TARP. Specific, non-limitingexamples of a conservative substitution include the following examples:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; LeuThe term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide. Non-conservative substitutions are thosethat reduce an activity or antigenicity.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Cancer: A malignant neoplasm that has undergone characteristic anaplasiawith loss of differentiation, increase rate of growth, invasion ofsurrounding tissue, and is capable of metastasis. For example, prostatecancer is a malignant neoplasm that arises in or from prostate tissue,and breast cancer is a malignant neoplasm that arises in or from breasttissue (such as a ductal carcinoma). Residual cancer is cancer thatremains in a subject after any form of treatment given to the subject toreduce or eradicate thyroid cancer. Metastatic cancer is a cancer at oneor more sites in the body other than the site of origin of the original(primary) cancer from which the metastatic cancer is derived.

CD4: Cluster of differentiation factor 4, a T cell surface protein thatmediates interaction with the MHC Class II molecule. CD4 also serves asthe primary receptor site for HIV on T cells during HIV infection. Cellsthat express CD4 are often helper T cells.

CD8: Cluster of differentiation factor 8, a T cell surface protein thatmediates interaction with the MHC Class I molecule. Cells that expressCD8 are often cytotoxic T cells.

Chemotherapy; chemotherapeutic agents: As used herein, any chemicalagent with therapeutic usefulness in the treatment of diseasescharacterized by abnormal cell growth. Such diseases include tumors,neoplasms and cancer as well as diseases characterized by hyperplasticgrowth such as psoriasis. In one embodiment, a chemotherapeutic agent isan agent of use in treating neoplasms such as solid tumors. In oneembodiment, a chemotherapeutic agent is a radioactive molecule. One ofskill in the art can readily identify a chemotherapeutic agent of use(e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 inHarrison's Principles of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., © 2000Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology PocketGuide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; FischerD S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993). The immunogenic TARPpolypeptides disclosed herein can be used in conjunction with additionalchemotherapeutic agents.

Degenerate variant: A polynucleotide encoding an epitope of TARP thatincludes a sequence that is degenerate as a result of the genetic code.There are 20 natural amino acids, most of which are specified by morethan one codon. Therefore, all degenerate nucleotide sequences areincluded in this disclosure as long as the amino acid sequence of theTARP polypeptide encoded by the nucleotide sequence is unchanged.

Dendritic cell (DC): Dendritic cells are the principle antigenpresenting cells (APCs) involved in primary immune responses. Dendriticcells include plasmacytoid dendritic cells and myeloid dendritic cells.Their major function is to obtain antigen in tissues, migrate tolymphoid organs and present the antigen in order to activate T cells.Immature dendritic cells originate in the bone marrow and reside in theperiphery as immature cells.

Diagnostic: Identifying the presence or nature of a pathologiccondition, such as, but not limited to, prostate cancer. Diagnosticmethods differ in their sensitivity and specificity. The “sensitivity”of a diagnostic assay is the percentage of diseased individuals who testpositive (percent of true positives). The “specificity” of a diagnosticassay is 1 minus the false positive rate, where the false positive rateis defined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis. “Prognostic” is theprobability of development (e.g., severity) of a pathologic condition,such as prostate cancer, or metastasis.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5, about 9, or 8 to 10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., “Epitope Mapping Protocols” in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). In oneembodiment, an epitope binds an MHC molecule, such an HLA molecule or aDR molecule. These molecules bind polypeptides having the correct anchoramino acids separated by about eight to about ten amino acids, such asnine amino acids.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus, expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see e.g., Bitter etal., Methods in Enzymology 153:516-544, 1987). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques can also be used to provide fortranscription of the nucleic acid sequences.

Heterologous: Originating from separate genetic sources or species. Apolypeptide that is heterologous to TARP originates from a nucleic acidthat does not encode TARP. In one specific, non-limiting example, apolypeptide comprising nine consecutive amino acids from TARP and aheterologous amino acid sequence includes a β-galactosidase, a maltosebinding protein, and albumin, or an immunoglobulin amino acid sequence.Generally, an antibody that specifically binds to a protein of interestwill not specifically bind to a heterologous protein.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4⁺ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence such that the peptide will bind an MHC molecule andinduce a cytotoxic T lymphocyte (“CTL”) response, or a B cell response(e.g. antibody production) against the antigen from which theimmunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide.

Immunogenic peptides can also be identified by measuring their bindingto a specific MHC protein (e.g. HLA-A02.01) and by their ability tostimulate CD4 and/or CD8 when presented in the context of the MHCprotein.

Immunogenic composition: A composition comprising an epitope of a TARPpolypeptide that induces a measurable CTL response against cellsexpressing TARP polypeptide, or induces a measurable B cell response(e.g., production of antibodies that specifically bind TARP) against aTARP polypeptide. It further refers to isolated nucleic acids encodingan immunogenic epitope of TARP polypeptide that can be used to expressthe epitope (and thus be used to elicit an immune response against thispolypeptide). For in vitro use, the immunogenic composition can consistof the isolated nucleic acid protein or peptide. For in vivo use, theimmunogenic composition will typically comprise the nucleic acid proteinor peptide in pharmaceutically acceptable carriers, and/or other agents.A TARP polypeptide, or nucleic acid encoding the polypeptide, can bereadily tested for its ability to induce a CTL by art-recognized assays.

Inhibiting or treating a disease: Inhibiting a disease, such as tumorgrowth, refers to inhibiting the full development of a disease. Inseveral examples, inhibiting a disease refers to lessening symptoms of atumor, such as preventing the development of paraneoplastic syndrome ina person who is known to have prostate or breast cancer, or lessening asign or symptom of the tumor. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition related to the disease, such as the tumor.

Isolated: An “isolated” biological component (such as a nucleic acid orprotein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, enzymatic linkages, and radioactive isotopes.

Linker sequence: A linker sequence is an amino acid sequence thatcovalently links two polypeptide domains. Linker sequences can beincluded in the between the TARP epitopes disclosed herein to providerotational freedom to the linked polypeptide domains and thereby topromote proper domain folding and presentation to the MHC. By way ofexample, in a recombinant polypeptide comprising two TARP-29-37-9V (SEQID NO: 6) domains, linker sequences can be provided between them, suchas a polypeptide comprising TARP-29-37-9V (SEQ ID NO:6)-linker-TARP-29-37-9V (SEQ ID NO: 6). Linker sequences, which aregenerally between 2 and 25 amino acids in length, are well known in theart and include, but are not limited to, the glycine(4)-serine spacer(GGGGS x3) described by Chaudhary et al., Nature 339:394-397, 1989.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Neoplasm: An abnormal cellular proliferation, which includes benign andmalignant tumors, as well as other proliferative disorders.

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Peptide: A chain of amino acids of between 3 and 30 amino acids inlength. In one embodiment, a peptide is from about 7 to about 25 aminoacids in length. In yet another embodiment, a peptide is from about 8 toabout 10 amino acids in length. In yet another embodiment, a peptide isabout 9 amino acids in length.

Peptide Modifications: TARP epitopes include synthetic embodiments ofpeptides described herein. In addition, analogs (non-peptide organicmolecules), derivatives (chemically functionalized peptide moleculesobtained starting with the disclosed peptide sequences) and variants(homologs) of these proteins can be utilized in the methods describedherein. Each polypeptide of this disclosure is comprised of a sequenceof amino acids, which may be either L- and/or D-amino acids, naturallyoccurring and otherwise.

Peptides can be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpeptides, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, can be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the peptide,whether amino-terminal or side chain, can be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or can be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into the peptides of thisinvention to select and provide conformational constraints to thestructure that result in enhanced stability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe peptide backbone and component amino acid side chains, resulting insuch peptido- and organomimetics of an immunogenic TARP polypeptidehaving measurable or enhanced ability to generate an immune response.For computer modeling applications, a pharmacophore is an idealizedthree-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs,” in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology, Munson (ed.) 1995, Ch. 102, for descriptionsof techniques used in CADD. Also included are mimetics prepared usingsuch techniques.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975),describes compositions and formulations suitable for pharmaceuticaldelivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

A “therapeutically effective amount” is a quantity of a chemicalcomposition or a cell to achieve a desired effect in a subject beingtreated. For instance, this can be the amount necessary to inhibit tumorgrowth or to measurably alter outward symptoms of the tumor. Whenadministered to a subject, a dosage will generally be used that willachieve target tissue concentrations (for example, in lymphocytes) thathas been shown to achieve an in vitro effect.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). In one embodiment, the polypeptide is a TARPpolypeptide.

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or reporter molecule. Primers are short nucleicacids, preferably DNA oligonucleotides, of about 15 nucleotides or morein length. Primers may be annealed to a complementary target DNA strandby nucleic acid hybridization to form a hybrid between the primer andthe target DNA strand, and then extended along the target DNA strand bya DNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, for example by polymerase chain reaction (PCR) orother nucleic-acid amplification methods known in the art. One of skillin the art will appreciate that the specificity of a particular probe orprimer increases with its length. Thus, for example, a primer comprising20 consecutive nucleotides will anneal to a target with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers can be selectedthat comprise about 20, 25, 30, 35, 40, 50 or more consecutivenucleotides.

Promoter: A promoter is an array of nucleic acid control sequences thatdirects transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, such as inthe case of a polymerase II type promoter (a TATA element). A promoteralso optionally includes distal enhancer or repressor elements which canbe located as much as several thousand base pairs from the start site oftranscription. Both constitutive and inducible promoters are included(see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987).

Specific, non-limiting examples of promoters include promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the retrovirus long terminal repeat; theadenovirus late promoter; the vaccinia virus 7.5K promoter) can be used.Promoters produced by recombinant DNA or synthetic techniques can alsobe used. A polynucleotide can be inserted into an expression vector thatcontains a promoter sequence which facilitates the efficienttranscription of the inserted genetic sequence of the host. Theexpression vector typically contains an origin of replication, apromoter, as well as specific nucleic acid sequences that allowphenotypic selection of the transformed cells.

Prostate Cancer: A malignant tumor, generally of glandular origin, ofthe prostate. Prostate cancers include adenocarcinomas and small cellcarcinomas. Many prostate cancers express prostate specific antigen(PSA).

Protein Purification: The epitopes of TARP disclosed herein can bepurified (and/or synthesized) by any of the means known in the art (see,e.g., Guide to Protein Purification, ed. Deutscher, Meth. Enzymol. 185,Academic Press, San Diego, 1990; and Scopes, Protein Purification:Principles and Practice, Springer Verlag, New York, 1982). Substantialpurification denotes purification from other proteins or cellularcomponents. A substantially purified protein is at least about 60%, 70%,80%, 90%, 95%, 98% or 99% pure. Thus, in one specific, non-limitingexample, a substantially purified protein is 90% free of other proteinsor cellular components.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified nucleicacid is one in which the nucleic acid is more enriched than the nucleicacid in its natural environment within a cell. Similarly, a purifiedpeptide preparation is one in which the peptide or protein is moreenriched than the peptide or protein is in its natural environmentwithin a cell. In one embodiment, a preparation is purified such thatthe protein or peptide represents at least about 60% (such as, but notlimited to, 70%, 80%, 90%, 95%, 98% or 99%) of the total peptide orprotein content of the preparation.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Selectively hybridize: Hybridization under moderately or highlystringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA versus DNA) of thehybridizing regions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

A specific example of progressively higher stringency conditions is asfollows: 2×SSC/0.1% SDS at about room temperature (hybridizationconditions); 0.2×SSC/0.1% SDS at about room temperature (low stringencyconditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringencyconditions); and 0.1×SSC at about 68° C. (high stringency conditions).One of skill in the art can readily determine variations on theseconditions (e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Washing can be carried out using only one ofthese conditions, e.g., high stringency conditions, or each of theconditions can be used, e.g., for 10-15 minutes each, in the orderlisted above, repeating any or all of the steps listed. However, asmentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a TARP polypeptide will possess a relativelyhigh degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Higgins and Sharp, Gene 73:237, 1988; Higginsand Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a TARP polypeptide are typically characterizedby possession of at least 75%, for example at least 80%, sequenceidentity counted over the full length alignment with the amino acidsequence of TARP using the NCBI Blast 2.0, gapped blastp set to defaultparameters. For comparisons of amino acid sequences of greater thanabout 30 amino acids, the Blast 2 sequences function is employed usingthe default BLOSUM62 matrix set to default parameters, (gap existencecost of 11, and a per residue gap cost of 1). When aligning shortpeptides (fewer than around 30 amino acids), the alignment should beperformed using the Blast 2 sequences function, employing the PAM30matrix set to default parameters (open gap 9, extension gap 1penalties). Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs andvariants will typically possess at least 80% sequence identity overshort windows of 10-20 amino acids, and can possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are available at the NCBI website on the internet.One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is entirely possible thatstrongly significant homologs could be obtained that fall outside of theranges provided.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a TARP specific binding agent is an agent thatbinds substantially to a TARP polypeptide. In one embodiment, thespecific binding agent is a monoclonal or polyclonal antibody thatspecifically binds TARP.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and veterinary subjects, including human andnon-human mammals.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 Tcell is a cytotoxic T lymphocyte. In another embodiment, a CD8 cell is asuppressor T cell.

T cell receptor γ Alternate Reading frame Protein (TARP): A polypeptidethat is translated from a form of the T cell receptor γ gene, which istranscribed in prostate cells of epithelial origin, in prostate cancercells, and in many breast cancers. TARP is disclosed in published PCTApplication No. WO 01/04309, published Jan. 18, 2001 (Pastan et al.),which is incorporated herein by reference.

In one embodiment, the polypeptide has a sequence set forth as:

-   -   MQMFPPSPLF FFLQLLKQSS RRLEHTF VFL RNFSLMLLRY IGKKRRATRF WDPRRGTP        (SEQ ID NO: 1, see also GENBANK® Accession No. AAG29337, which        is herein incorporated by reference).

In other embodiments, TARP has an amino acid sequence least 90%identical to SEQ ID NO: 1, for example a polypeptide that has about 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or even higher sequence identity toSEQ ID NO: 1. Additional variants have been described (see below and seepublished PCT Application No. WO 01/04309 herein incorporated byreference, for a complete description of these polypeptides).

In another embodiment, TARP is encoded by nucleic acid having a sequenceset forth as:

gggcaagagt tgggcaaaaa aatcaaggta tttggtcccg gaacaaagct tatcattacagataaacaac ttgatgcaga tgtttccccc aagcccacta tttttcttcc ttcaattgctgaaacaaagc tccagaaggc tggaacatac ctttgtcttc ttgagaaatt tttccctgatgttattaaga tacattggca agaaaagaag agcaacacga ttctgggatc ccaggaggggaacaccatga agactaacga cacatacatg aaatttagct ggttaacggt gccagaaaagtcactggaca aagaacacag atgtatcgtc agacatgaga ataataaaaa cggagttgatcaagaaatta tctttcctcc aataaagacg gatgtcatca caatggatcc caaagacaattgttcaaaag atgcaaatga tacactactg ctgcagctca caaacacctc tgcatattacatgtacctcc tcctgctcct caagagtgtg gtctattttg ccatcatcac ctgctgtctgcttagaagaa cggctttctg ctgcaatgga gagaaatcat aacagacggt ggcacaaggaggccatcttt tcctcatcgg ttattgtccc tagaagcgtc ttctgaggat ctagttgggctttctttctg ggtttgggcc atttcagttc tcatgtgtgt actattctat cattattgtataacggtttt caaaccagtg ggcacacaga gaacctcact ctgtaataac aatgaggaatagccacggcg atctccagca ccaatctctc catgttttcc acagctcctc cagccaacccaaatagcgcc tgctatagtg tagacatcct gcggcttcta gccttgtccc tctcttagtgttctttaatc agataactgc ctggaagcct ttcattttac acgccctgaa gcagtcttctttgctagttg aattatgtgg tgtgtttttc cgtaataagc aaaataaatt taaaaaaatgaaaagtt(SEQ ID NO: 2, see also GENBANK® Accession No. AF151103,which is hereinincorporated by reference).See also Wolfgang et al., Proc. Natl. Acad. Sci. U.S.A. 97(17):9437-9442, 2000, and WO 01/04309, published Jan. 18, 2001 (Pastanet al.), which is incorporated herein by reference.

Therapeutically active polypeptide: An agent, such as an epitope of TARPthat causes induction of an immune response, as measured by clinicalresponse (for example increase in a population of immune cells,increased cytolytic activity against cells that express TARP, ormeasurable reduction of tumor burden). Therapeutically active moleculescan also be made from nucleic acids. Examples of a nucleic acid basedtherapeutically active molecule is a nucleic acid sequence that encodesa TARP epitope, wherein the nucleic acid sequence is operably linked toa control element such as a promoter.

In one embodiment, a therapeutically effective amount of an epitope ofTARP is an amount used to generate an immune response, or to treatprostate cancer or breast cancer in a subject. Specific, non-limitingexamples are a polypeptide having a sequence set forth as SEQ ID NOs:3-7. Treatment refers to a therapeutic intervention that ameliorates asign or symptom of prostate cancer or breast cancer, or a reduction intumor burden.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergene and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Immunogenic TARP Peptides

T cell receptor γ Alternate Reading frame Protein (TARP) is apolypeptide that is translated from a form of the T cell receptor γgene, which is transcribed in prostate cells of epithelial origin, inprostate cancer cells, and in many breast cancers. TARP is disclosed inpublished PCT Application No. WO 01/04309, published Jan. 18, 2001(Pastan et al.), which is incorporated herein by reference.

In one embodiment, the polypeptide has a sequence set forth as:

-   -   MQMFPPSPLF FFLQLLKQSS RRLEHTF VFL RNFSLMLLRY IGKKRRATRF WDPRRGTP        (SEQ ID NO: 1, see also GenBank Accession No. AAG29337, which is        herein incorporated by reference).

In other embodiments, TARP has an amino acid sequence least 90%identical to SEQ ID NO: 1, for example a polypeptide that has about 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or even higher sequence identity toSEQ ID NO: 1. Additional variants have been described (see below and seepublished PCT Application No. WO 01/04309 herein incorporated byreference, for a complete description of these polypeptides).

In another embodiment, TARP is encoded by a nucleic acid having asequence set forth as:

gggcaagagt tgggcaaaaa aatcaaggta tttggtcccg gaacaaagct tatcattacagataaacaac ttgatgcaga tgtttccccc aagcccacta tttttcttcc ttcaattgctgaaacaaagc tccagaaggc tggaacatac ctttgtcttc ttgagaaatt tttccctgatgttattaaga tacattggca agaaaagaag agcaacacga ttctgggatc ccaggaggggaacaccatga agactaacga cacatacatg aaatttagct ggttaacggt gccagaaaagtcactggaca aagaacacag atgtatcgtc agacatgaga ataataaaaa cggagttgatcaagaaatta tctttcctcc aataaagacg gatgtcatca caatggatcc caaagacaattgttcaaaag atgcaaatga tacactactg ctgcagctca caaacacctc tgcatattacatgtacctcc tcctgctcct caagagtgtg gtctattttg ccatcatcac ctgctgtctgcttagaagaa cggctttctg ctgcaatgga gagaaatcat aacagacggt ggcacaaggaggccatcttt tcctcatcgg ttattgtccc tagaagcgtc ttctgaggat ctagttgggctttctttctg ggtttgggcc atttcagttc tcatgtgtgt actattctat cattattgtataacggtttt caaaccagtg ggcacacaga gaacctcact ctgtaataac aatgaggaatagccacggcg atctccagca ccaatctctc catgttttcc acagctcctc cagccaacccaaatagcgcc tgctatagtg tagacatcct gcggcttcta gccttgtccc tctcttagtgttctttaatc agataactgc ctggaagcct ttcattttac acgccctgaa gcagtcttctttgctagttg aattatgtgg tgtgtttttc cgtaataagc aaaataaatt taaaaaaatgaaaagtt(SEQ ID NO: 2, see also GenBank Accession No. AF151103, which is hereinincorporated by reference). See also Wolfgang et al., Proc. Natl. Acad.Sci. U.S.A. 97(17):9437-9442, 2000, and WO 01/04309, published Jan. 18,2001 (Pastan et al.), which is incorporated herein by reference.Immunogenic fragments of TARP and TARP itself, can also be chemicallysynthesized by standard methods. If desired, polypeptides can also bechemically synthesized by emerging technologies. One such process isdescribed in W. Lu et al., Federation of European Biochemical SocietiesLetters. 429:31-35, 1998. Polypeptides can also be produced usingmolecular genetic techniques, such as by inserting a nucleic acidencoding TARP or an epitope thereof into an expression vector,introducing the expression vector into a host cell, and isolating thepolypeptide (see below).

Immunogenic TARP polypeptides are disclosed herein. These peptidesinclude nine consecutive amino acids from the consensus sequence:

-   -   FVF LX₁NFSLMX₂ (SEQ ID NO: 9), wherein X₁ is R or A, and wherein        X₂ is L or V.

SEQ ID NO: 9 is equivalent to amino acids 27 to 37 of SEQ ID NO: 1,wherein amino acid 31 is an R or an A and wherein amino acid 37 is an Lor a V. It should be note that in SEQ ID NO: 1, amino acid 31 is an Rand amino acid 37 is a L. In one specific, non-limiting example, X₁ isan A and X₂ is an L. In other specific non-limiting examples, X₁ is Aand X₂ is V, X₁ is R and X₂ is L, or X₁ is R and X₂ is V.

The immunogenic TARP polypeptides disclosed herein does not include allthe additional consecutive amino acids of TARP (SEQ ID NO: 1). In oneembodiment, the polypeptide does not include amino acids 1-26 of TARP(SEQ ID NO: 1), and the polypeptide does not include amino acids 38-58of TARP (SEQ ID NO: 1).

It is believed that the presentation of peptides by MHC Class Imolecules involves binding to the cleft in an MHC Class I moleculethrough the anchor residues of the peptide and ultimate presentation onthe cell surface. Depending upon the particular anchor residues, amongother things, certain peptides can bind more tightly to particular HLAmolecules than others. Peptides that bind well are usually “dominant”epitopes, while those that bind less well are often “subdominant” or“cryptic” epitopes. Dominant epitopes of either self proteins or foreignproteins evoke strong tolerance or immune responses. Subdominant orcryptic epitopes generate weak responses or no responses at all. Withoutbeing bound by theory, tighter binding by dominant epitopes to HLAmolecules results in their denser presentation on the cell surface,greater opportunity to react with immune cells and greater likelihood ofeliciting an immune response or tolerance. MHC Class I molecules presentepitopes from endogenous proteins for presentation to CTL cells. HLA A,HLA B and HLA C molecules bind peptides of about 8 to 10 amino acids inlength that have particular anchoring residues. The anchoring residuesrecognized by an HLA Class I molecule depend upon the particular allelicform of the HLA molecule. A CD8+ T cell bears T cell receptors thatrecognize a specific epitope when presented by a particular HLA moleculeon a cell. When a CTL precursor that has been stimulated by an antigenpresenting cell to become a cytotoxic T lymphocyte contacts a cell thatbears such an HLA-peptide complex, the CTL forms a conjugate with thecell and destroys it. In several examples presented herein, thepolypeptides that are disclosed bind and are presented by HLA-A2.1.

In one specific, non-limiting example, an immunogenic TARP polypeptideincludes one of the following amino acid sequences:

FLRNFSLML (TARP-29-37, SEQ ID NO: 3) FVFLRNFSL (TARP-27-35, SEQ ID NO:4) FLANFSLML (TARP-29-37-3A, SEQ ID NO: 5) FLRNFSLMV, (TARP-29-37-9V,SEQ ID NO: 6)but does not include additional TARP sequences, such as a additionalepitope included in SEQ ID NO: 1. In one example, the polypeptideconsists of the amino acids sequence set forth as SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, or SEQ ID NO:6, and do not include additional aminoacids. In another specific, non-limiting example, the polypeptide doesnot include consecutive amino acids 1-26 of SEQ ID NO: 1 or consecutiveamino acids 38-58 of SEQ ID NO: 1.

In another example, the polypeptide can also include heterologoussequences to TARP (e.g. amino acid sequences of at least nine aminoacids in length that are not included in SEQ ID NO: 1). Thus, in severalspecific non-limiting examples, the immunogenic peptide is a fusionpolypeptide, for example the polypeptide includes six sequentialhistidine residues, a β-galactosidase amino acid sequence, or aimmunoglobulin amino acid sequence. The polypeptide can also becovalently linked to a carrier.

The polypeptide can optionally include repetitions of any one of SEQ IDNOs: 3-6. In one specific, non-limiting example, the polypeptideincludes 2, 3, 4, 5, or up to ten repetitions of one of the sequencesset forth as SEQ ID NOs: 3-6. A linker sequence can optionally beincluded between the repetitions.

In another embodiment, the polypeptide consists of one of the followingamino acid sequences:

FLRNFSLML (TARP-29-37, SEQ ID NO: 3) FVFLRNFSL (TARP-27-35, SEQ ID NO:4) FLANFSLML (TARP-29-37-3A, SEQ ID NO: 5) FLRNFSLMV. (TARP-29-37-9V,SEQ ID NO: 6)

The immunogenic TARP polypeptides disclosed herein can also bechemically synthesized by standard methods, or can be producedrecombinantly. An exemplary process for polypeptide production isdescribed in Lu et al., Federation of European Biochemical SocietiesLetters. 429:31-35, 1998. They can also be isolated by methods includingpreparative chromatography and immunological separations.

In other embodiments, fusion proteins are provided comprising a firstand second polypeptide moiety in which one of the protein moietiescomprises an amino acid sequence of at least five amino acidsidentifying an epitope of TARP, such as a polypeptide described by SEQID NO: 9. In several examples, the TARP moiety is any one of, SEQ ID NO:3-6. The other moiety can be a carrier protein and/or an immunogenicprotein. Such fusions also are useful to evoke an immune responseagainst TARP.

A TARP polypeptide can be covalently linked to a carrier, which is animmunogenic macromolecule to which an antigenic molecule can be bound.When bound to a carrier, the bound polypeptide becomes more immunogenic.Carriers are chosen to increase the immunogenicity of the bound moleculeand/or to elicit higher titers of antibodies against the carrier whichare diagnostically, analytically, and/or therapeutically beneficial.Covalent linking of a molecule to a carrier can confer enhancedimmunogenicity and T cell dependence (see Pozsgay et al., PNAS96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18, 1976; Dintzis etal., PNAS 73:3671-75, 1976). Useful carriers include polymeric carriers,which can be natural (for example, polysaccharides, polypeptides orproteins from bacteria or viruses), semi-synthetic or syntheticmaterials containing one or more functional groups to which a reactantmoiety can be attached. Bacterial products and viral proteins (such ashepatitis B surface antigen and core antigen) can also be used ascarriers, as well as proteins from higher organisms such as keyholelimpet hemocyanin, horseshoe crab hemocyanin, edestin, mammalian serumalbumins, and mammalian immunoglobulins. Additional bacterial productsfor use as carriers include bacterial wall proteins and other products(for example, streptococcal or staphylococcal cell walls andlipopolysaccharide (LPS)).

Polynucleotides encoding the immunogenic TARP polypeptides disclosedherein are also provided. These polynucleotides include DNA, cDNA andRNA sequences which encode the polypeptide of interest.

A nucleic acid encoding an immunogenic TARP polypeptide can be cloned oramplified by in vitro methods, such as the polymerase chain reaction(PCR), the ligase chain reaction (LCR), the transcription-basedamplification system (TAS), the self-sustained sequence replicationsystem (3SR) and the Qβ replicase amplification system (QB). Forexample, a polynucleotide encoding the protein can be isolated bypolymerase chain reaction of cDNA using primers based on the DNAsequence of the molecule. A wide variety of cloning and in vitroamplification methodologies are well known to persons skilled in theart. PCR methods are described in, for example, U.S. Pat. No. 4,683,195;Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; andErlich, ed., PCR Technology, (Stockton Press, NY, 1989). Polynucleotidesalso can be isolated by screening genomic or cDNA libraries with probesselected from the sequences of the desired polynucleotide understringent hybridization conditions.

The polynucleotides encoding an immunogenic TARP polypeptide include arecombinant DNA which is incorporated into a vector into an autonomouslyreplicating plasmid or virus or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (e.g., a cDNA)independent of other sequences. The nucleotides of the invention can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single and double forms of DNA.

DNA sequences encoding an immunogenic TARP polypeptide can be expressedin vitro by DNA transfer into a suitable host cell. The cell may beprokaryotic or eukaryotic. The term also includes any progeny of thesubject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. Methods of stable transfer, meaning that the foreignDNA is continuously maintained in the host, are known in the art.

A polynucleotide sequences encoding an immunogenic TARP polypeptide canbe operatively linked to expression control sequences. An expressioncontrol sequence operatively linked to a coding sequence is ligated suchthat expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. The expression controlsequences include, but are not limited to, appropriate promoters,enhancers, transcription terminators, a start codon (i.e., ATG) in frontof a protein-encoding gene, splicing signal for introns, maintenance ofthe correct reading frame of that gene to permit proper translation ofmRNA, and stop codons.

The polynucleotide sequences encoding an immunogenic TARP polypeptidecan be inserted into an expression vector including, but not limited to,a plasmid, virus or other vehicle that can be manipulated to allowinsertion or incorporation of sequences and can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art.

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding an immunogenicTARP polypeptide, and a second foreign DNA molecule encoding aselectable phenotype, such as the herpes simplex thymidine kinase gene.Another method is to use a eukaryotic viral vector, such as simian virus40 (SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

Therapeutic Methods and Pharmaceutical Compositions

An immunogenic TARP polypeptide as disclosed herein can be administeredto a subject in order to generate an immune response. In one embodiment,a therapeutically effective amount of an immunogenic TARP polypeptidecomprising SEQ ID NO: 9, which is equivalent to amino acids 27 to 37 ofSEQ ID NO: 1, wherein amino acid 31 is an R or an A and wherein aminoacid 37 is an L or a V. It should be noted that in SEQ ID NO: 1, aminoacid 31 is an R and amino acid 37 is an L. In specific, non-limitingexamples, X₁ is A and X₂ is L, X₁ is A and X₂ is V, X₁ is R and X₂ is L,or X₁ is R and X₂ is V. The immunogenic TARP polypeptide does notinclude additional consecutive amino acids of TARP (SEQ ID NO: 1), suchthat the polypeptide does not include amino acids 1-26 of TARP (SEQ IDNO: 1), and the polypeptide does not include amino acids 38-58 of TARP(SEQ ID NO: 1).

In one specific, non-limiting example, an immunogenic TARP polypeptideis administered that includes one of the following amino acid sequences:

FLRNFSLML (TARP-29-37, SEQ ID NO: 3) FVFLRNFSL (TARP-27-35, SEQ ID NO:4) FLANFSLML (TARP-29-37-3A, SEQ ID NO: 5) FLRNFSLMV (TARP-29-37-9V, SEQID NO: 6)but does not include additional TARP sequences, such as an epitopeincluded in SEQ ID NO: 1 that is not SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, or SEQ ID NO: 6. Thus, the polypeptide does not include aminoacids 1-26 of SEQ ID NO: 1 or amino acids 38-58 of SEQ ID NO: 1. One ormore of these immunogenic TARP polypeptides can be administered to asubject to treat prostate or breast cancer. Thus, one, two, three or allfour of these immunogenic TARP polypeptides can be administered to asubject.

In exemplary applications, compositions are administered to a patientsuffering from a disease, such as prostate or breast cancer, in anamount sufficient to raise an immune response to TARP-expressing cells.Administration induces a sufficient immune response to slow theproliferation of such cells or to inhibit their growth, or to reduce asign or a symptom of the tumor. Amounts effective for this use willdepend upon the severity of the disease, the general state of thepatient's health, and the robustness of the patient's immune system. Atherapeutically effective amount of the compound is that which provideseither subjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer.

An immunogenic TARP polypeptide can be administered by any means knownto one of skill in the art (see Banga, A., “Parenteral ControlledDelivery of Therapeutic Peptides and Proteins,” in Therapeutic Peptidesand Proteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995) suchas by intramuscular, subcutaneous, or intravenous injection, but evenoral, nasal, or anal administration is contemplated. In one embodiment,administration is by subcutaneous or intramuscular injection. To extendthe time during which the peptide or protein is available to stimulate aresponse, the peptide or protein can be provided as an implant, an oilyinjection, or as a particulate system. The particulate system can be amicroparticle, a microcapsule, a microsphere, a nanocapsule, or similarparticle. (see, e.g., Banga, supra). A particulate carrier based on asynthetic polymer has been shown to act as an adjuvant to enhance theimmune response, in addition to providing a controlled release. Aluminumsalts can also be used as adjuvants to produce an immune response.

In one specific, non-limiting example, an immunogenic TARP polypeptideis administered in a manner to direct the immune response to a cellularresponse (that is, a cytotoxic T lymphocyte (CTL) response), rather thana humoral (antibody) response. A number of means for inducing cellularresponses, both in vitro and in vivo, are known. Lipids have beenidentified as agents capable of assisting in priming CTL in vivo againstvarious antigens. For example, as described in U.S. Pat. No. 5,662,907,palmitic acid residues can be attached to the alpha and epsilon aminogroups of a lysine residue and then linked (e.g., via one or morelinking residues, such as glycine, glycine-glycine, serine,serine-serine, or the like) to an immunogenic peptide. The lipidatedpeptide can then be injected directly in a micellar form, incorporatedin a liposome, or emulsified in an adjuvant. As another example, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine canbe used to prime tumor specific CTL when covalently attached to anappropriate peptide (see, Deres et al., Nature 342:561, 1989). Further,as the induction of neutralizing antibodies can also be primed with thesame molecule conjugated to a peptide which displays an appropriateepitope, two compositions can be combined to elicit both humoral andcell-mediated responses where that is deemed desirable.

In yet another embodiment, to induce a CTL response to an immunogenicTARP polypeptide, a MHC Class II-restricted T-helper epitope is added tothe immunogenic TARP polypeptide to induce T-helper cells to secretecytokines in the microenvironment to activate CTL precursor cells. Thetechnique further involves adding short lipid molecules to retain theconstruct at the site of the injection for several days to localize theantigen at the site of the injection and enhance its proximity todendritic cells or other “professional” antigen presenting cells over aperiod of time (see Chesnut et al., “Design and Testing of Peptide-BasedCytotoxic T-Cell-Mediated Immunotherapeutics to Treat InfectiousDiseases and Cancer,” in Powell et al., eds., Vaccine Design, theSubunit and Adjuvant Approach, Plenum Press, New York, 1995).

A pharmaceutical composition including an immunogenic TARP polypeptideis thus provided. In one embodiment, the immunogenic TARP polypeptide,or fragment thereof, is mixed with an adjuvant containing two or more ofa stabilizing detergent, a micelle-forming agent, and an oil. Suitablestabilizing detergents, micelle-forming agents, and oils are detailed inU.S. Pat. Nos. 5,585,103; 5,709,860; 5,270,202; and 5,695,770, all ofwhich are incorporated by reference. A stabilizing detergent is anydetergent that allows the components of the emulsion to remain as astable emulsion. Such detergents include polysorbate, 80 (TWEEN)(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured byICI Americas, Wilmington, Del.), TWEEN 40™, TWEEN 20™, TWEEN 60™,Zwittergent™ 3-12, TEEPOL HB7™, and SPAN 85™. These detergents areusually provided in an amount of approximately 0.05 to 0.5%, such as atabout 0.2%. A micelle forming agent is an agent which is able tostabilize the emulsion formed with the other components such that amicelle-like structure is formed. Such agents generally cause someirritation at the site of injection in order to recruit macrophages toenhance the cellular response. Examples of such agents include polymersurfactants described by BASF Wyandotte publications, e.g., Schmolka, J.Am. Oil. Chem. Soc. 54:110, 1977, and Hunter et al., J. Immunol129:1244, 1981, PLURONIC™ L62LF, L101, and L64, PEG1000, and TETRONIC™1501, 150R1, 701, 901, 1301, and 130R1. The chemical structures of suchagents are well known in the art. In one embodiment, the agent is chosento have a hydrophile-lipophile balance (HLB) of between 0 and 2, asdefined by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent canbe provided in an effective amount, for example between 0.5 and 10%, orin an amount between 1.25 and 5%.

The oil included in the composition is chosen to promote the retentionof the antigen in oil-in-water emulsion, i.e., to provide a vehicle forthe desired antigen, and preferably has a melting temperature of lessthan 65° C. such that emulsion is formed either at room temperature(about 20° C. to 25° C.), or once the temperature of the emulsion isbrought down to room temperature. Examples of such oils includesqualene, Squalane, EICOSANE™, tetratetracontane, glycerol, and peanutoil or other vegetable oils. In one specific, non-limiting example, theoil is provided in an amount between 1 and 10%, or between 2.5 and 5%.The oil should be both biodegradable and biocompatible so that the bodycan break down the oil over time, and so that no adverse affects, suchas granulomas, are evident upon use of the oil.

An adjuvant can be included in the composition. In one embodiment, theadjuvant is a mixture of stabilizing detergents, micelle-forming agent,and oil available under the name Provax® (IDEC Pharmaceuticals, SanDiego, Calif.). An adjuvant can also be an immunostimulatory nucleicacid, such as a nucleic acid including a CpG motif.

In another embodiment, a pharmaceutical composition includes a nucleicacid encoding an immunogenic TARP polypeptide or immunogenic fragmentthereof. A therapeutically effective amount of the immunogenic TARPpolynucleotide can be administered to a subject in order to generate animmune response. In one specific, non-limiting example, atherapeutically effective amount of the immunogenic TARP polynucleotideis administered to a subject to treat prostate cancer or breast cancer.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Asdescribed above, the nucleotide sequence encoding an immunogenic TARPpolypeptide can be placed under the control of a promoter to increaseexpression of the molecule.

Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response), and U.S. Pat. Nos.5,593,972 and 5,817,637 (which describe operably linking a nucleic acidsequence encoding an antigen to regulatory sequences enablingexpression). U.S. Pat. No. 5,880,103 describes several methods ofdelivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™, negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A™ (saponin). Protective immunity has beengenerated in a variety of experimental models of infection, includingtoxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ asthe delivery vehicle for antigens (Mowat and Donachie, Immunol. Today12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS™have been found to produce Class I mediated CTL responses (Takahashi etal., Nature 344:873, 1990).

In another approach to using nucleic acids for immunization, animmunogenic TARP polypeptide can also be expressed by attenuated viralhosts or vectors or bacterial vectors. Recombinant vaccinia virus,adeno-associated virus (AAV), herpes virus, retrovirus, or other viralvectors can be used to express the peptide or protein, thereby elicitinga CTL response. For example, vaccinia vectors and methods useful inimmunization protocols are described in U.S. Pat. No. 4,722,848. BCG(Bacillus Calmette Guerin) provides another vector for expression of thepeptides (see Stover, Nature 351:456-460, 1991).

In one embodiment, a nucleic acid encoding an immunogenic TARPpolypeptide is introduced directly into cells. For example, the nucleicacid can be loaded onto gold microspheres by standard methods andintroduced into the skin by a device such as Bio-Rad's Helios™ Gene Gun.The nucleic acids can be “naked,” consisting of plasmids under controlof a strong promoter. Typically, the DNA is injected into muscle,although it can also be injected directly into other sites, includingtissues in proximity to metastases. Dosages for injection are usuallyaround 0.5 μg/kg to about 50 mg/kg, and typically are about 0.005 mg/kgto about 5 mg/kg (see, e.g., U.S. Pat. No. 5,589,466).

In one specific, non-limiting example, a pharmaceutical composition forintravenous administration would include about 0.1 μg to 10 mg ofimmunogenic TARP polypeptide per patient per day. Dosages from 0.1 up toabout 100 mg per patient per day can be used, particularly if the agentis administered to a secluded site and not into the circulatory or lymphsystem, such as into a body cavity or into a lumen of an organ. Actualmethods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remingtons Phamaceutical Sciences, 19^(th) Ed.,Mack Publishing Company, Easton, Pa., 1995.

The compositions can be administered for therapeutic treatments. Intherapeutic applications, a therapeutically effective amount of thecomposition is administered to a subject suffering from a disease, suchas prostate or breast cancer. Single or multiple administrations of thecompositions are administered depending on the dosage and frequency asrequired and tolerated by the subject. In one embodiment, the dosage isadministered once as a bolus, but in another embodiment can be appliedperiodically until a therapeutic result is achieved. Generally, the doseis sufficient to treat or ameliorate symptoms or signs of diseasewithout producing unacceptable toxicity to the subject. Systemic orlocal administration can be utilized.

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems, see Banga, Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., 1995. Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein as acentral core. In microspheres, the therapeutic agent is dispersedthroughout the particle. Particles, microspheres, and microcapsulessmaller than about 1 μm are generally referred to as nanoparticles,nanospheres, and nanocapsules, respectively. Capillaries have a diameterof approximately 5 μm so that only nanoparticles are administeredintravenously. Microparticles are typically around 100 μm in diameterand are administered subcutaneously or intramuscularly (see Kreuter,Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc.,New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on ControlledDrug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y.,pp. 315-339, 1992).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., Int. J. Pharm. 112:215,1994). In yet another aspect, liposomes are used for controlled releaseas well as drug targeting of the lipid-capsulated drug (Betageri et al.,Liposome Drug Delivery Systems, Technomic Publishing Co., Inc.,Lancaster, Pa., 1993). Numerous additional systems for controlleddelivery of therapeutic proteins are known (e.g., U.S. Pat. Nos.5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; and5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697;4,902,505; 5,506,206; 5,271,961; 5,254,342; and 5,534,496).

In another method, antigen presenting cells (APCs), such as dendriticcells, are pulsed or co-incubated with peptides comprising animmunogenic TARP polypeptide in vitro. In one specific, non-limitingexample, the antigen presenting cells can be autologous cells. Atherapeutically effective amount of the antigen presenting cells canthen be administered to a subject.

The immunogenic TAP polypeptide can be delivered to the dendritic cellsor to dendritic cell precursors via any method known in the art,including, but not limited to, pulsing dendritic cells directly withantigen, or utilizing a broad variety of antigen delivery vehicles, suchas, for example, liposomes, or other vectors known to deliver antigen tocells. In one specific, non-limiting example an antigenic formulationincludes about 0.1 μg to about 1,000 μg, or about 1 to about 100 μg of aselected immunogenic TARP polypeptide. The immunogenic TARP polypeptidecan also be administered with agents that promote dendritic cellmaturation. Specific, non-limiting examples of agents of use areinterleukin-4 (IL-4) and granulocyte/macrophage colony stimulatingfactor (GM-CSF), or flt-3 ligand (flt-3L). The preparation can alsocontain buffers, excipients, and preservatives, amongst otheringredients.

In one embodiment, mature antigen presenting cells are generated topresent the immunogenic TARP polypeptide. These dendritic cells are thenadministered alone to a subject with a tumor that expresses TARP, suchas a prostate or a breast cancer. In another embodiment, the maturedendritic cells are administered in conjunction with a chemotherapeuticagent.

Alternatively, the APCs are used to sensitize CD8 cells, such as tumorinfiltrating lymphocytes (TILs) from prostate or breast tumors orperipheral blood lymphocytes (PBLs). The TILs or PBLs can be from thesame subject (autologous) that is to be treated. Alternatively, the TILsor PBLs can be heterologous. However, they should at least be MHCClass-I restricted to the HLA types the subject possesses. An effectiveamount of the sensitized cells are then administered to the subject.

Peripheral blood mononuclear cells (PBMCs) can be used as the respondercell source of CTL precursors. The appropriate antigen-presenting cellsare incubated with peptide, after which the peptide-loadedantigen-presenting cells are then incubated with the responder cellpopulation under optimized culture conditions. Positive CTL activationcan be determined by assaying the culture for the presence of CTLs thatkill radio-labeled target cells, both specific peptide-pulsed targets aswell as target cells expressing endogenously processed forms of theantigen from which the peptide sequence was derived, such as TARP (e.g.SEQ ID NO: 1).

The cells can be administered to a subject to inhibit the growth ofcells of TARP expressing tumors. In these applications, atherapeutically effective amount of activated antigen presenting cells,or activated lymphocytes, are administered to a subject suffering from adisease, in an amount sufficient to raise an immune response toTARP-expressing cells. The resulting immune response is sufficient toslow the proliferation of such cells or to inhibit their growth, or toreduce a sign or a symptom of the tumor.

In a supplemental method, any of these immunotherapies is augmented byadministering a cytokine, such as IL-2, IL-3, IL-6, IL-10, IL-12, IL-15,GM-CSF, interferons.

In a further method, any of these immunotherapies is augmented byadministering an additional chemotherapeutic agent. In one example, thisadministration is sequential. Examples of such agents are alkylatingagents, antimetabolites, natural products, or hormones and theirantagonists. Examples of alkylating agents include nitrogen mustards(such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard orchlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (suchas carmustine, lomustine, semustine, streptozocin, or dacarbazine).Examples of antimetabolites include folic acid analogs (such asmethotrexate), pyrimidine analogs (such as 5-FU or cytarabine), andpurine analogs, such as mercaptopurine or thioguanine. Examples ofnatural products include vinca alkaloids (such as vinblastine,vincristine, or vindesine), epipodophyllotoxins (such as etoposide orteniposide), antibiotics (such as dactinomycin, daunorubicin,doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (suchas L-asparaginase). Examples of miscellaneous agents include platinumcoordination complexes (such as cis-diamine-dichloroplatinum II alsoknown as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocroticalsuppressants (such as mitotane and aminoglutethimide). Examples ofhormones and antagonists include adrenocorticosteroids (such asprednisone), progestins (such as hydroxyprogesterone caproate,medroxyprogesterone acetate, and magestrol acetate), estrogens (such asdiethylstilbestrol and ethinyl estradiol), antiestrogens (such astamoxifen), and androgens (such as testerone proprionate andfluoxymesterone). Examples of the most commonly used chemotherapy drugsinclude Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU,Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU,Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin,Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, suchas docetaxel), Velban, Vincristine, VP-16, while some more newer drugsinclude Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11),Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin),Xeloda (Capecitabine), Zevelin and calcitriol. Non-limiting examples ofimmunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.),bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocytemacrophage colony stimulating factor; Genetics Institute), IL-2 (Cetusor Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG(from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosisfactor; Genentech).

Reagents for the Detection of Cells that Express CD8 (CD8+) Cells thatSpecifically Bind TARP

Reagents are provided herein for the detection of CD8 expressing cellsthat specifically bind TARP. These reagents are tetrameric MHC ClassI/immunogenic TARP polypeptide complexes. These tetrameric complexesinclude an immunogenic TARP polypeptide that includes nine consecutiveamino acids from the consensus sequence:

-   -   FVF LX₁NFSLMX₂ (SEQ ID NO: 9), wherein X₁ is R or A, and wherein        X₂ is L or V.

SEQ ID NO: 9 is equivalent to amino acids 27 to 37 of SEQ ID NO: 1,wherein amino acid 31 is an R or an A and wherein amino acid 37 is an Lor a V. It should be note that in SEQ ID NO: 1, amino acid 31 is an Rand amino acid 37 is a L. In one specific, non-limiting example, X₁ isan A and X₂ is an L. In other specific non-limiting examples, X₁ is Aand X₂ is V, X₁ is R and X₂ is L, or X₁ is R and X₂ is V.

The tetrameric complexes disclosed herein do not include additionalconsecutive amino acids of TARP (SEQ ID NO: 1), such that thepolypeptide does not include amino acids 1-26 of TARP (SEQ ID NO: 1),and the polypeptide does not include amino acids 38-58 of TARP (SEQ IDNO: 1).

In several examples, the tetrameric complex includes an immunogenic TARPpolypeptide that includes one of the following amino acid sequences:

FLRNFSLML (TARP-29-37, SEQ ID NO: 3) FVFLRNFSL (TARP-27-35, SEQ ID NO:4) FLANFSLML (TARP-29-37-3A, SEQ ID NO: 5) FLRNFSLMV, (TARP-29-37-9V,SEQ ID NO: 6)but does not include additional TARP sequences, such as an epitopeincluded in SEQ ID NO: 1 that is not SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, or SEQ ID NO: 6.

Tetrameric MHC Class I/peptide complexes can be synthesized usingmethods well known in the art (Altmann et al., Science 274:94, 1996,which is herein incorporated by reference). In one specific non-limitingexample, purified HLA heavy chain and β2-microglobulin (β2m) can besynthesized by means of a prokaryotic expression system. One specific,non-limiting example of an expression system of use is the pET system(R&D Systems, Minneapolis, Minn.). The heavy chain is modified bydeletion of the trans-membrane and cytosolic tail and COOH-terminaladdition of a sequence containing the biotin protein ligase (Bir-A)enzymatic biotinylation site. Heavy chain, β2m, and peptide are thenrefolded. The refolded product can be isolated by any means known in theart, and then biotinylated by Bir-A. A tetramer is then produced bycontacting the biotinylated product with strepavidin.

In one embodiment, the strepavidin is labeled. Suitable labels include,but are not limited to, enzymes, magnetic beads, colloidal magneticbeads, haptens, fluorochromes, metal compounds, radioactive compounds ordrugs. The enzymes that can be conjugated to strepavidin include, butare not limited to, alkaline phosphatase, peroxidase, urease andβ-galactosidase. The fluorochromes that can be conjugated to thestrepavidin include, but are not limited to, fluorescein isothiocyanate,tetramethylrhodamine isothiocyanate, phycoerythrin, allophycocyanins andTEXAS RED®. For additional fluorochromes that can be conjugated tostrepavidin, see Haugland, R. P., Molecular Probes: Handbook ofFluorescent Probes and Research Chemicals (1992-1994). The metalCompounds that can be conjugated to the strepavidin include, but are notlimited to, ferritin, colloidal gold, and particularly, colloidalsuperparamagnetic beads. The haptens that can be conjugated to thestrepavidin include, but are not limited to, biotin, digoxigenin,oxazalone, and nitrophenol. The radioactive compounds that can beconjugated to strepavidin are known to the art, and include but are notlimited to technetium 99m (⁹⁹Tc), ¹²⁵I and amino acids comprising anyradionuclides, including, but not limited to, ¹⁴C, ³H and ³⁵S.Generally, strepavidin labeled with a fluorochrome is utilized in themethods disclosed herein.

In one embodiment, suspension of cells including T cells thatspecifically recognize TARP is produced, and the cells are reacted withthe tetramer in suspension. In one embodiment, these reagents are usedto label cells, which are then analyzed by fluorescence activated cellsorting (FACS). A machine for FACS employs a plurality of colorchannels, low angle and obtuse light-scattering detection channels, andimpedance channels, among other more sophisticated levels of detection,to separate or sort cells. Any FACS technique can be employed as long asit is not detrimental to the detection of the desired cells. (Forexemplary methods of FACS see U.S. Pat. No. 5,061,620.)

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

To develop more effective immunotherapy for the cancer patients, it isimportant to find new tumor-specific antigens and establishing newvaccine strategies are important. Vasmatzis et al. (Proc Natl Acad SciUSA 95:300, 1998) have recently found new genes specifically expressedin human prostate by expressed sequence tag database analysis, and oneof them, TCRγ alternate reading frame protein (TARP), is expressed inboth prostate and breast cancer cell lines (Wolfgang et al., Proc NatlAcad Sci USA 97:943, 2000). This new protein, TARP, originates fromepithelial cells and is located in the nucleus. Further study (Wolfganget al., Cancer Res 61:8122, 2001) showed that TARP is expressed in theandrogen-sensitive prostate cancer cells, LNCaP but not inandrogen-independent PC3 cell line. TARP also has a role in regulatinggrowth and gene expression in prostate cancer cell lines.

As disclosed herein, TARP is expressed in normal prostate and in mostprostate cancers. Furthermore, TARP is expressed in anandrogen-sensitive cell line, LNCaP, but not in the androgen-independentPC3 cell line, which suggests that TARP plays a role in prostate cancerprogression (Wolfgang et al., Cancer Res 61:8122, 2001). Treatment ofLNCaP cells with testosterone resulted in the increased expression ofTARP in LNCaP cells. This protein is also expressed in a broad range ofbreast cancer cell lines, MCF-7, SK-BR3, BT-474, and CRL1897 (Wolfganget al., Proc Natl Acad Sci USA 97:9437, 2000).

As disclosed herein, it was determined that human HLA-A2-presentedepitopes derived from the TARP, and then their immunogenicities weretested in A2K^(b) transgenic mice that have a chimeric MHC Class Imolecules composed of α1 and α2 domains from HLA-A2.1 and an α3 domainfrom K^(b) (Sherman et al., Science 258:81, 1992). Next, theimmunogenicity of the peptides was enhanced by increasing their bindingaffinities to HLA-A2.1 molecules, and the result showed that enhancedepitopes were more immunogenic, which could increase the efficacy of avaccine (e.g., see Berzofsky et al., Ann. N.Y. Acad. Sci. 690:256,1993). As it takes a stronger signal to activate a response than to bethe target of a response (Alexander et al., J. Exp. Med. 173:849, 1991),the natural epitope may be sufficient to allow killing of the tumor by Tcells raised with the enhanced epitope. Furthermore, CD8⁺ T cellsreactive to the wild-type and enhanced epitopes were detected inprostate cancer patients. By in vitro restimulation of PBMC fromprostate cancer patients with peptide-loaded dendritic cells (DC),peptide-specific CD8⁺ T cells were detected and expanded that recognizedpeptide/MHC complexes and killed a human breast cancer cell line. Inaddition, HLA-A2.1-tetramers composed of individual peptides made inthis study provide a method by which the presence of peptide-specificCD8⁺ T cells has been detected in the patients.

Example 1 Materials and Methods

Animals. A2K^(b) transgenic mice expressing a chimeric HLA-A2.1transgene with the α1 and α2 domains from HLA-A2.1 and the α3 domainfrom H2K^(b), to allow binding to mouse CD8, on a C57/BL6 backgroundhave been described (Sherman et al., Science 258:81, 1992). These micewere bred and housed in appropriate animal care facilities. Allprocedures with animals were conducted in accordance with theinstitutionally approved protocols.

Peptides. HLA-A2.1-binding peptides were synthesized on a Model Symphonypeptide synthesizer (Perkin-Elmer, Boston, Mass.) using conventionalf-MOC chemistry and cleaved from the resin by trifluoroacetic acid. Thepurity and molar concentration were analyzed by reverse-phase HPLC on aC18 column using a gradient of 0.1% trifluoroacetic acid in water and0.1% trifluoroacetic acid in acetonitrile, and were further purified bypreparative reverse phase HPLC using a similar gradient. Full-lengthpeptides were purchased from Multiple Peptide Systems (San Diego,Calif.) at >95% purity and were single peaks by reverse-phase HPLC.

Cell lines. The T2 cell line is deficient in TAP1 and TAP2 transporterproteins and expresses low levels of HLA-A2.1. C1R-A2.1 cell line ishuman B lymphoblastoid cell line HMYC1R transfected with HLA-A2.1. Cellswere maintained in complete medium (RPMI-1640 supplemented with 10% FCS,100 IU penicillin, and 10 μg/ml streptomycin). RPMI 1640 and othersupplements were purchased from Cellgro (Gaithersberg, Md.). ForC1R-A2.1 cells, 200 μg/ml geneticin (Sigma, St. Louis, Mo.) was addedinto the medium. LNCaP, PC3, MCF-7, and DU145 cells were maintained incomplete media. For PC3-TARP cells (Wolfgang et al., Cancer Res 61:8122,2001), 200 μg/ml hygromycin B (Invitrogen, Carlsbad, Calif.) was addedinto the medium.

T2-binding assay. Peptide binding capacity to HLA-A2.1 molecules wasmeasured by using the T2 cell line according to a protocol previouslydescribed (Nijman et al., Eu. J. Immunol. 23:1215, 1993). T2 cells(3×10⁵/well) were incubated overnight in 96-well plates with culturemedium (1:1 mixture of RPMI 1640/EHAA containing 2.5% FBS, 100 U/mlpenicillin, 100 μg/ml streptomycin) with 10 μg/ml β2-microglobulin(Sigma) and different concentrations of peptide. Cells were washed twicewith cold PBS containing 2% FBS and incubated for 30 minutes at 4° C.with anti-HLA-A2.1 BB7.2 mAb (1/80 dilution from hybridoma supernatant).After washing, cells were stained with FITC-labeled goat anti-mouse Ig(PharMingen, San Diego, Calif.) and the level of HLA-A2.1 expression wasmeasured by flow cytometry. HLA-A2.1 expression was quantified asfluorescence index (FI) according to the formula: FI=(mean fluorescenceintensity with peptide-mean fluorescence intensity without peptide)/meanfluorescence intensity without peptide.

Immunizations. A2K^(b) transgenic mice were immunized with syngeneicpeptide-loaded dendritic cells (DC), plasmid DNA expressing TARP, or themixture of peptide and cytokine in incomplete Freund's adjuvant (IFA).For DC immunization, DC were pulsed with 10 μM peptide in serum-freeRPMI for 2 hours at 37° C., and then mice were immunized subcutaneously(s.c.) with 1-3×10⁵ DC without washing. DC were prepared from bonemarrow as previously described (Celluzzi et al. J. Exp. Med. 183:283,1996). Alternatively, mice were immunized intramuscularly with 100 μgplasmid DNA, pcDNA5/FRT/TARP, generated by inserting the TARP DNA intothe pcDNA5/RFT (Invitrogen, Carlsbad, Calif.) vector. Mice were alsoimmunized subcutaneously (s.c.) in the base of the tail with 100 μl ofan emulsion containing 1:1 IFA and PBS with antigens and cytokines (50nmol CTL epitope, 50 nmol HBV core 128-140 helper epitope, 3 μg ofIL-12, and 5 μg of granulocyte macrophage colony stimulating factor(GM-CSF). IFA and cytokines were purchased from Sigma and Peptotech(Rocky Hill, N.J.), respectively.

In vitro human CD8⁺ T cell priming with DC. Elutriated monocytes andlymphocytes were obtained from HLA-A2.1-positive patients or normaldonors. To prepare DC, monocytes were cultured at 10⁶ cells/ml incomplete medium containing human IL-4 (1000 units/ml) and human GM-CSF(1000 units/ml). On day 2 and 4, half of the media was exchanged. Tomature the DC, CD40 ligand trimer (Immunex, Seattle, Wash.) was added at1 μg/ml on day 4 or 5, and then further cultured for 2 or 3 days. Cellswere harvested on day 7, and then pulsed with 10 μM of peptides for 2hours before γ-irradiation. 1×10⁵ peptide-pulsed DC and 2×10⁶ autologousCD8⁺ T cells were mixed and cultured in 24-well plates. 10 units/ml IL-2was added on day 2 during restimulation. Cells were restimulated every7-9 days approximately 4-7 cycles.

CTL assay. For murine CTL, CD8⁺ T cells from the immunized mice wererestimulated with peptide-loaded splenocytes for one week as previouslydescribed (Oh et al., J Immunol 170:2523, 2003), and then applied to 5hour ⁵¹Cr release assays. Target cells were labeled with ⁵¹Cr first andwashed twice. Cells were then pulsed with peptides for 2 hours and usedas target cells without further washing. For human CTL, target cellswere also labeled with ⁵¹Cr first, and then loaded with peptides. In theCTL assay against human tumor cells, target cells were incubated in thecomplete medium containing 1000 units/ml IFN-γ for 72 hours. The mean oftriplicate samples was calculated, and the percentage of specific lysiswas determined using the following formula: Percentage of specificlysis=100×[(experimental ⁵¹Cr release−control ⁵¹Cr release)/(maximum⁵¹Cr release−control ⁵¹Cr release)]. The maximum release refers tocounts from targets in 2.5% Triton X-100.

HLA-A2.1 tetramers. Tetrameric MHC Class I/peptide complexes weresynthesized as described (Altmann et al., Science 274:94, 1996).Briefly, purified HLA heavy chain and β2-microglobulin (β2m) weresynthesized by means of a prokaryotic expression system (pET; R&DSystems, Minneapolis, Minn.). The heavy chain was modified by deletionof the trans-membrane and cytosolic tail and COOH-terminal addition of asequence containing the Bir-A enzymatic biotinylation site. Heavy chain,β2m, and peptide were refolded by dilution. The refolded product wasisolated by FPLC and then biotinylated by Bir-A in the presence ofbiotin, adenosine 5′-triphosphate, and Mg⁺⁺ (all from Sigma).Streptavidin-PE conjugate (PharMingen) was added in 1:4 molar ratio.

Antibody and flow cytometry. FITC-labeled anti-mouse CD8 (53-6.7),CD11c, CD80 (B7-1), CD54 (ICAM-1), anti-human CD8 (RPA-T8), CD14 (5E2),CD80 (B7-1), and CD86 (B7-2) were used for staining of cell surfacemolecules. For intracellular IFN-γ staining, cells were stained byfollowing the manufacturer's protocol. All Abs and reagents werepurchased from PharMingen. For flow cytometric analysis of cell surface,5×10⁵ cells were washed and resuspended in PBS containing 0.2% BSA and0.1% sodium azide. Cells were incubated on ice with the appropriateantibody for 30 minutes and then washed. Samples were analyzed on aFACScan (BD Biosciences, Mountain View, Calif.). Background staining wasassessed by use of an isotype control antibody. For tetramer staining,cells were incubated with FITC-labeled anti-CD8 for 10 minutes, and thenstained with tetramers.

Example 2 HLA-A2.1-Restricted Epitope Prediction and Wild-Type PeptideBinding Affinity to HLA-A2.1 Molecules

Fifty-eight amino acid residues in the TARP protein were previouslycharacterized (Wolfgang et al., Proc Natl Acad Sci USA 97:9437, 2000)(FIG. 1A). To determine HLA-A2.1 epitopes from the TARP, four differentwild-type peptides were first predicted based on the anchor residues(FIG. 1B) and their binding affinities to HLA-A2.1 molecules weremeasured by the T2-binding assay. As shown in FIG. 1C, only twowild-type peptides, TARP-29-37 and TARP-27-35, showed measurable bindingcapacity to HLA-A2.1 molecules. Although both peptides overlap by sevenresidues, TARP-27-35 had almost a ten-fold higher binding affinity toHLA-A2.1 molecules than TARP-29-37. Neither TARP-2-9 nor TARP-22-30showed any measurable binding affinity to HLA-A2.1 molecules.

The theoretical half-life of peptide binding to HLA-A2.1 molecules wasalso predicted by running the software program for peptide motif search(Parker et al., J. Immunol. 152:163, 1994), and the results wereconsistent with the data in FIG. 1C.

TARP is composed of 58 amino acid residues and contains severalhydrophobic amino acids including five leucines, but data from theT2-binding assay showed that only two wild-type peptides (TARP-29-37,SEQ ID NO: 3 and TARP-27-35, SEQ ID NO: 4) had a measurable bindingaffinity to HLA-A2.1 molecules. Seven out of nine amino acids in bothwild-type peptides overlap. Moreover, they both share two amino acids atsame positions, Phe at position 1 and Leu at position 9, although theamino acid at position 1 is not a primary anchor residue. However,TARP-27-35 (SEQ ID NO: 4) showed a better binding affinity thanTARP-29-37 (SEQ ID NO: 3). Without being bound by theory, this isprobably because of other amino acids in non-anchor positions, such asPhe at position 3 in TARP-27-35 (SEQ ID NO: 4). Although the other twowild-type peptides, TARP-2-9 (SEQ ID NO: 7) and TARP-22-30 (SEQ ID NO:8), possess Leu at position 9 and Met or Leu at position 2,respectively, neither TARP-2-9 (SEQ ID NO: 7) nor TARP-22-30 (SEQ ID NO:8) showed a measurable binding affinity to HLA-A2.1 molecules. NeitherTARP-27-35 (SEQ ID NO: 4) and TARP-29-37 (SEQ ID NO: 3) have anyresidues known to be associated with poor HLA-A2.1 binding at thesecondary anchor positions. Without being bound by theory, this suggeststhat primary anchor residues alone are not sufficient to determine thebinding affinity of peptides (Ruppert et al., Cell 74:929, 1993;Rammensee et al., Immunogenetics 41:178, 1994), and/or the large numberof Pro residues affected the conformation of TARP-2-9 (SEQ ID NO: 7) andthat the Glu at position 3 may have interfered with binding ofTARP-22-30 (SEQ ID NO: 8). Although epitope enhancement by replacing theGlu in TARP-22-30 (SEQ ID NO: 8) might have improved binding, if thewild-type binding is too weak to serve as a good target for CTL raisedagainst the enhanced-peptide, it is unlikely that such anenhanced-peptide would be useful.

Example 3 Wild-Type HLA-A2.1 Epitopes Induce Peptide-Specific CD8⁺ TCell Responses in A2K^(b) Transgenic Mice

To verify whether those two wild-type peptides predicted in FIG. 1B areimmunogenic or not, A2K^(b) transgenic mice were immunized with eitherpeptide-pulsed DC or plasmid DNA expressing the TARP. As shown in FIGS.2A and 2B, both peptides could induce peptide-specific CD8⁺ T cellresponses in the mice immunized by either immunization protocol, butresponses were higher after peptide-pulsed DC immunization. Compared toTARP-27-35, however, TARP-29-37 resulted in lower peptide-specific CD8⁺T cell responses, indicating that binding affinity of peptide to MHCmolecules is a major factor that regulates the induction of CD8⁺ T cellresponses. The number of IFN-γ-producing CD8⁺ T cells was also measuredby intracellular staining. Consistent with the CTL data, a greaternumber of CD8⁺ T cells, 2.1% of total CD8⁺ T cells, was obtained fromthe mice immunized with DC pulsed with TARP-27-35 than the miceimmunized with TARP-29-37-pulsed DC. However, the data indicated thatmice immunized with TARP-29-37 pulsed-DC also had a significant numberof CD8⁺ T cells producing IFN-γ. The number of IFN-γ-producing CD8⁺ Tcells was also measured in mice immunized with DNA plasmid and the datawere consistent with the data in FIG. 2C.

Data from the DNA immunization experiments indicate that the murineantigen processing system is not a limitation for production of peptidespresented by human Class I molecules. Moreover, the data suggests thatsuch HLA-transgenic mice can be used for the study of peptidesrecognized by CD8⁺ T cells specific for HLA-A2/peptide complex.Peptide/HLA-A2 complexes recognized by murine T cells might be differentfrom those recognized by human T cells (explaining why thecross-reactivity of CD8⁺ T cells from mouse and human was not exactlythe same). It is theoretically possible that those species differencesmay be due to different TCR repertoires. However, the data showed thatboth wild-type and enhanced epitopes resulted in the induction ofpeptide-specific CD8 T cells in HLA-A2 transgenic mice, and thereforethese mice can be used as good predictors of human T cell epitopes.

Example 4 Amino Acid Substitutions in the Wild-Type Peptides Result inIncreased Binding Affinity to HLA-A2 Molecules (Epitope-Enhancement)

Binding affinity of peptide to MHC Class I molecules is a major factordetermining the immunogenicity of peptide epitopes. To enhance thebinding affinity of wild-type epitopes, TARP-29-37 (SEQ ID NO: 3) andTARP-27-35 (SEQ ID NO: 4), amino acids in the peptides were substitutedwith others. For TARP-29-37, Arg at position 3 and Leu at position 9were substituted with Ala (TARP-29-37-3A, SEQ ID NO: 5) and Val(TARP-29-37-9V, SEQ ID NO: 5), respectively (FIG. 3A). As shown in FIG.3C, substitution at position 3 with Ala in TARP-29-37 resulted in thegreatest increase in the binding affinity of the peptide. The bindingaffinity of TARP-29-37-3A was not less than that of the positive controlpeptide, FMP (flu matrix peptide) (Gotch et al., Nature 326:881, 1987).Although TARP-29-37-9V (SEQ ID NO: 6) showed a lower binding affinity toHLA-A2.1 than TARP-29-37-3A (SEQ ID NO: 5) did, substitution of Leu atposition 9 with Val did enhance the binding affinity of wild-typepeptide, TARP-29-37 (SEQ ID NO: 3). In addition to TARP-29-37 (SEQ IDNO: 3), it was tried to improve the binding affinity of TARP-27-35 (SEQID NO: 4) by substitution of amino acids in position 2, 3, and 9 withLeu, Ala, and Val, respectively, but there was no significantenhancement in binding affinity to HLA-A2.1 molecules. In contrast tothe situation with TARP-29-37, substitution with Ala at position 3 inTARP-27-35 (SEQ ID NO: 4, see FIG. 3B) resulted in no binding of thepeptide to HLA-A2.1 molecules, suggesting that the peptide bindingaffinity to MHC molecules was not simply determined by a single aminoacid, but influenced by other amino acids in the epitope. Two othersubstitutions at position 2 of TARP-27-35 (SEQ ID NO: 4) with Leu(TARP-27-35-2L) and at position 9 of TARP-27-35 (SEQ ID NO: 4) with Val(TARP-27-35-9V) did not alter the binding affinity of the wild-typepeptide, TARP-27-35 (SEQ ID NO: 4) (FIG. 3B).

As disclosed herein, when a wild-type peptide does bind, but perhaps notoptimally, one strategy to improve the usage of self-peptides such asTARP-29-37 (SEQ ID NO: 3) and TARP-27-35 (SEQ ID NO: 4) by developingenhanced-epitopes that are potentially more immunogenic. Substitution ofArg at position 3 with Ala in TARP-29-37 (SEQ ID NO: 3) greatly improvedthe peptide binding affinity to the HLA-A2.1 molecules. This could beexplained by an adverse effect of Arg at position 3 reducing the peptidebinding affinity to HLA-A2.1 as well as the stability of the peptide/MHCcomplexes. Substitution of Leu at position 9 with Val in TARP-29-37 (SEQID NO: 3) also resulted in the increased binding affinity of thepeptide.

Substitutions were also made in TARP-27-35 (SEQ ID NO: 4) bysubstitutions for Val at position 2, Phe at position 3, and Leu atposition 9 with Leu, Ala, and Val, respectively, but those substitutionsdid not improve the binding affinity of TARP-27-35 (SEQ ID NO: 4). Aminoacid residues associated with poor binding to HLA-A2.1 are Asp, Glu, andPro at position 1; Asp and Glu at position 3; Arg, Lys, His, and Ala atposition 4; Pro at position 5; Arg, Lys, and His at position 7; Asp,Glu, Arg, Lys, and His at position 8; and Arg, Lys, and His at position9 (Ruppert et al., Cell 74:929, 1993; Rammensee et al., Immunogenetics41:178, 1995). Both TARP-27-35 and TARP-29-37 do not have any residueknown to be associated with poor HLA-A2 binding at the secondary anchorpositions. In contrast, as noted, the low binding affinity of thewild-type peptides, TARP₂₋₉ and TARP-22-30, is probably due to the Proat position 5 and Glu at position 3, respectively.

Example 5 Immunogenicity of the Enhanced Epitopes and CD8⁺ T CellResponses to the Wild-Type Peptides

Both TARP-29-37-3A (SEQ ID NO: 5) and TARP-29-37-9V (SEQ ID NO: 6)showed better binding affinity to HLA-A2.1 molecules than the wild-type,TARP-29-37 (SEQ ID NO: 3) (see FIGS. 1 and 3). To test theimmunogenicities and recognition of the wild-type peptide by CD8⁺ Tcells induced with the enhanced epitopes, mice were immunized with thosepeptides. As shown in FIG. 4A, both TARP-29-37-3A (SEQ ID NO: 5) andTARP-29-37-9V (SEQ ID NO: 6) induced almost two-fold higher frequenciesof CD8⁺ T cells specific for individual peptides compared to thewild-type, TARP-29-37 (SEQ ID NO: 3). Another wild-type epitope,TARP-27-35 (SEQ ID NO: 4) also induced a higher frequency of CD8⁺ CTLspecific for the peptide (but these CD8⁺ CTLs did not recognizeTARP-29-37 (SEQ ID NO: 3) and the epitope-enhanced peptides, see FIG.4B).

In a cross-reactivity analysis (see FIGS. 4B-D), CD8⁺ CTL induced withboth enhanced epitopes lysed target cells pulsed with TARP-29-37 (SEQ IDNO: 3), suggesting that the TCRs of those CTL recognize the wild-typepeptide (TARP-29-37)-MHC Class I complex to some extent. Theimmunogenicity of the enhanced versus wild-type TARP-29-37 peptidesshown in FIG. 4A correlated with the peptide affinity for HLA-A2.1(shown in FIG. 3). However, CD8⁺ CTL induced with TARP-29-37-3A (SEQ IDNO: 5) recognized TARP-29-37-3A/MHC complex better than otherpeptide/MHC Class I complexes. In contrast, CTL raised againstTARP-29-37-9V (SEQ ID NO: 6) were slightly more potent in killingtargets pulsed with wild-type TARP-29-37 (SEQ ID NO: 3), suggesting thatthey could be more effective against tumor cells expressing the naturalantigen (FIG. 4B). When compared quantitatively (FIGS. 4C and 4D),TARP-29-37-9V (SEQ ID NO: 6) was the most immunogenic of the peptides ininducing CTLs specific for the wild-type sequence. Thus, TARP-29-37-9V(SEQ ID NO: 6) could be the most potent of the immunogens for inducingCTLs against tumor cells expressing the natural antigen.

Interestingly, CD8⁺ CTL induced with TARP-29-37-9V (SEQ ID NO: 6) couldalso kill target cells pulsed with TARP-27-35 (SEQ ID NO: 4). Incontrast, CTL induced with TARP-29-37-3A (SEQ ID NO: 5) did not killtarget cells pulsed with TARP-27-35 (SEQ ID NO: 4), suggesting that CD8⁺T cells induced with TARP-29-37-9V (SEQ ID NO: 6) have broadercross-reactivity to wild-type peptide/MHC complexes than CD8⁺ T cellsinduced with TARP-29-37-3A (SEQ ID NO: 5). This suggests thatTARP-29-37-9V (SEQ ID NO: 6) can be used to induce CD8⁺ T cells thatcould recognize antigen expressed on tumor cells.

Example 6 Human CD8⁺ T Cells Raised In Vitro Lyse Peptide-Pulsed TargetCells

TARP expression is particularly high in the prostate of prostate cancerpatients, and human prostate cancer cell lines also express high levelof TARP (e.g., see Essand et al., Proc Natl Acad Sci USA 96:9287, 1999;Wolfgang et al., Proc Natl Acad Sci USA 97:9437, 2000; Wolfgang et al.,Cancer Res 61:8122, 2001).

To test for the presence of peptide-specific CD8⁺ T cells (FIG. 5A) inan HLA-A2.1-positive prostate cancer patient, CD8⁺ T cells from theleukapheresis of the patient donor were restimulated with peptide-pulsedautologous DC in several cycles. During the in vitro restimulation,peptide-specific CD8⁺ T cells were first observed from the CD8⁺ T cellculture restimulated with TARP-29-37-3A (SEQ ID NO: 5) pulsed-DC, andthen TARP-29-37-9V (SEQ ID NO: 6) specific CD8⁺ T cells were raised.During the in vitro restimulation, peptide-specific CD8⁺ T cells weredetected after only four cycles of restimulation with TARP-29-37-3A (SEQID NO: 5)-pulsed DCs, whereas TARP-29-37-9V (SEQ ID NO: 6)-specific CD8⁺T cells required at least five cycles.

For both wild-type peptides (TARP-29-37 (SEQ ID NO: 3) and TARP-27-35(SEQ ID NO: 4), CD8⁺ CTL required at least 6 cycles of in vitrorestimulation to be detected. Cytolytic activity of those CD8⁺ CTLraised with individual peptides was tested against peptide-pulsedC1R-A2.1 target cells. All four CD8⁺ CTL could lyse peptide-pulsedtarget cells specifically, as shown in FIG. 5B. However, CD8⁺ CTL raisedwith TARP-29-37-3A (SEQ ID NO: 5) and TARP-27-35 (SEQ ID NO: 4) resultedin higher levels of cytolytic activity against the correspondingpeptides compared to CD8⁺ CTL raised with TARP-29-37 (SEQ ID NO: 3) andTARP-29-37-9V (SEQ ID NO: 6).

Example 7 Human CD8⁺ T Cells Raised Against TARP-29-37-9V (SEQ ID NO:6), but not TARP-29-37-3A (SEQ ID NO: 5), Recognize the MHC Complex withthe Wild-Type Peptide, TARP-29-37 (SEQ ID NO: 3)

As disclosed herein, murine CD8⁺ CTL induced by the enhanced epitopeslysed wild-type peptide-pulsed target cells (FIG. 4) and human CD8⁺ CTLraised against individual peptides lysed target cells pulsed with thecorresponding peptides (FIG. 5). However, it is important to knowwhether human CD8⁺ CTL raised against enhanced epitopes could lysetarget cells pulsed with wild-type peptide expected to be presented ontumor cells. To address this question, cytolytic activity of individualCD8⁺ CTL was measured against target cells pulsed with differentpeptides. As shown in FIG. 6A, human CD8⁺ CTL raised with TARP-29-37(SEQ ID NO: 3) could recognize and lyse target cells pulsed with thewild-type as well as the two enhanced epitopes (TARP-29-37-3A (SEQ IDNO: 5) and TARP-29-37-9V (SEQ ID NO: 6). However, TARP-29-37-3A-(SEQ IDNO: 5) specific CD8⁺ T cells poorly recognized the wild-type andTARP-29-37-9V (SEQ ID NO: 6), although they recognized TARP-29-37-3A(SEQ ID NO: 5) very well.

Unlike CD8⁺ T cells raised with TARP-29-37-3A (SEQ ID NO: 5), CD8⁺ Tcells specific for TARP-29-37-9V (SEQ ID NO: 6) could recognize bothwild-type and TARP-29-37-3A comparably. Data from FIG. 6 confirm thatCD8⁺ T cells raised with TARP-29-37-9V (SEQ ID NO: 6), but not 29-37-3A(SEQ ID NO: 5), could recognize both wild-type and enhanced epitope/MHCcomplex. CD8⁺ CTL induced with TARP-27-35 (SEQ ID NO: 4) recognizedTARP-27-35 (SEQ ID NO: 4)/MHC complex, but poorly recognized otherpeptide/MHC complexes tested. The data also showed that all three CTLraised with TARP-29-37 (SEQ ID NO: 3), TARP-29-37-3A (SEQ ID NO: 5), andTARP-29-37-9V (SEQ ID NO: 6) could recognize TARP-27-35 (SEQ ID NO: 4)to some extent, possibly due to the 7-residue overlap between them.

The avidity of CD8⁺ CTLs specific for TARP-29-37-9V to differentpeptides was measured (FIG. 6B). CTLs for TARP-29-37-9V (SEQ ID NO: 6)recognized TARP-29-37-9V (SEQ ID NO: 6) at the 0.001 μM level. Althoughthe avidity of these CTLs for wild-type TARP-29-37 (SEQ ID NO: 3) waslower, they could recognize the wild-type as well as TARP-29-37-3A (SEQID NO: 5) with only slightly lower avidity. However, these CTLs did notrecognize TARP-27-35 (SEQ ID NO: 4) at <10 μM concentration.

Example 8 Human CD8⁺ T Cells Raised Against TARP-29-37-9V and TARP-27-35Kill TARP-Expressing Tumor Cells

As disclosed herein, CD8⁺ T cells specific for individual peptides lysethe target cells pulsed with corresponding peptides. In addition,TARP-29-37 (SEQ ID NO: 3)- and TARP-29-37-9V (SEQ ID NO: 6)-specificCD8⁺ T cells killed the wild-type peptide (TARP-29-37, SEQ ID NO:5)-pulsed target cells efficiently. CD8⁺ T cells specific for TARP-27-35(SEQ ID NO: 4) also lysed TARP-27-35-pulsed target cells. To testwhether those CTL could kill human tumor cells that endogenously expressTARP, a CTL assay was performed against tumor cell lines that expressboth HLA-A2.1 and TARP, and the data are shown in FIG. 7A. At a 50:1 E/Tratio, all CD8⁺ T cells could kill the breast cancer line, MCF-7, butshowed marginal (10-12%) lytic activity against the prostate cancer cellline, LNCaP. Before CTL assay, all target cells were cultured in mediumcontaining IFN-γ, and the expression levels of HLA-A2.1 before and afterIFN-γ-treatment were measured (FIG. 7B). As expected from the CTL assay,LNCaP cells express an extremely low level of HLA-A2.1 and the level ofHLA-A2.1 was not much increased by the culture of the cells in mediumcontaining IFN-γ. In contrast, the level of HLA-A2.1 in MCF-7 was higherto start and greatly enhanced by IFN-γ. Both control cell lines, DU-145and PC3-TARP did not express HLA-A2.1. Similar to the HLA-A2 expressionlevels, data from real-time PCR showed that IFN-γ did not increase theexpression level of TARP in LNCaP cells but slightly and variablyincreased the level in MCF-7 cells. Of four different CD8⁺ CTL, CD8⁺ Tcells specific for either TARP-29-37-9V (SEQ ID NO: 6) or TARP-27-35(SEQ ID NO: 4) showed higher lytic activity against MCF-7 than CD8⁺ CTLraised with TARP-29-37 (SEQ ID NO: 3) and TARP-29-37-3A (SEQ ID NO: 5).Although CD8⁺ CTL specific for TARP-29-37-3A (SEQ ID NO: 5) showed lesslytic activity to MCF-7 at a 50:1 E/T ratio than the other three CD8⁺CTL did, comparable range of lytic activity against MCF-7 cells wasobserved at high E/T ratio (100:1 E/T ratio).

CD8⁺ CTLs typically express clonally distributed TCRs that possessexquisite specificity for a particular MHC/peptide complex. In both miceand humans, however, CD8⁺ T cells raised with individual peptides canrecognize a range of cross-reactive peptide/MHC complexes, depending onthe individual CD8⁺ CTLs and peptides. The cross-reactivity observed inthis study not only among variants of one peptide but between the twowild-type peptides could be explained by the fact that the two wild-typepeptides overlap by seven amino acid residues. A number of recentstudies have shown degenerate recognition of MHC/peptide complexes byindividual TCRs: examples range from T-cell recognition of dissimilarpeptides presented by the same MHC molecules to recognition of identicalpeptides bound to different MHC molecules. For example, CD8⁺ T cellsspecific for one peptide of polyoma virus recognize another epitope thathas no sequence homology. However, those CD8⁺ T cells require a muchhigher concentration of the alternative peptide for recognition.

In the cross-reactivity test for human and CD8⁺ T cells, CD8⁺ T cellsspecific for TARP-29-37 (SEQ ID NO: 3) could recognize all fourpeptide/MHC complexes. However, CD8⁺ T cells specific for TARP-27-35 orTARP-29-37-3A (SEQ ID NO: 5) did not recognize other peptides as much asthey did the immunogens. Of the enhanced peptides, only CTLs specificfor TARP-29-37-9V (SEQ ID NO: 6) could recognize the wild-type peptide,TARP-29-37 (SEQ ID NO: 3), to a similar degree to the immunogen. Inconsideration of choosing peptides for immunotherapy, eitherTARP-29-37-9V (SEQ ID NO: 6) or TARP-27-35 (SEQ ID NO: 4) will likely bemore potent than the other two peptides. Although TARP-29-37-3A (SEQ IDNO: 5) showed the highest binding affinity and resulted in high CD8⁺ Tcell responses in transgenic mice (FIG. 4D), CD8⁺ T cells specific forthis peptide did not recognize wild-type peptide very well in the humanand showed weak cross-reactivity to wild-type peptide in the mice. Forthe tumor killing assay, CD8⁺ T cells specific for TARP-29-37-3A (SEQ IDNO: 5) showed a significant range of specific lysis only at a 100:1 E:Tratio. At a 50:1 E:T ratio, CD8⁺ T cells specific for TARP-29-37-3A (SEQID NO: 5) showed a much lower lytic activity against tumor cells thanCTLs to TARP-29-37-9V (SEQ ID NO: 6). In contrast, TARP-29-37-9V (SEQ IDNO: 6) induced a higher level of CD8⁺ T cell responses than TARP-29-37,and CD8⁺ T cells specific for TARP-29-37-9V (SEQ ID NO: 6) couldrecognize the wild-type peptide, TARP-29-37 (SEQ ID NO: 3), and couldkill human tumor cells. CD8⁺ T cells specific for TARP-27-35 (SEQ ID NO:4) could kill human tumor cells as well.

Example 9 HLA-A2.1-Tetramers with Individual Peptides RecognizePeptide-Specific CD8⁺ T Cells in the Patients

Data shown in FIGS. 5 to 7 indicate that prostate cancer patients haveCD8⁺ T cells that recognize individual peptide and HLA-A2.1 complexes.This is compatible with previously published data that the expressionlevel of TARP is significantly elevated in the prostate of prostatecancer patients (Wolfgang et al., Proc Natl Acad Sci USA 97:9437, 2000).To determine whether those CD8⁺ T cells exist only in the patient, CD8⁺T cells from normal donors were repeatedly stimulated withpeptide-pulsed autologous DC, but CD8⁺ T cells were not raised that werespecific for any of the tested peptides in this experiment. To examinethe frequency of peptide specific CD8⁺ T cells in the prostate cancerpatients, tetramers were made that were composed of individual peptidesbound to HLA-A2.1. PBMC were stained with anti-CD8 and these tetramers.As shown in FIG. 8, all four tetramers detected CD8⁺ T cells from theprostate cancer patients, but not the normal donors, suggesting thatthose tetramers could be used for detection of peptide-specific CD8⁺ Tcells, such as for diagnostic purposes. The results also indicate thatthe presence of prostate cancer is sufficient to induce CD8⁺ T cellsspecific for these epitopes to a fairly high frequency (as 0.6% to 3% oftotal CD8⁺ T cells in the patients' PBMC). The frequency ofpeptide-specific CD8⁺ T cells could be dependent on the stage of tumorin the patients.

In each case, the frequency of tetramer-positive cells was substantiallyhigher in the patient than in a normal donor tested concurrently. TCRrepertoire usage was analyzed in peptide-specific CD8⁺ T cells fromprostate cancer patient 1, and the data indicate that CD8⁺ T cellsspecific for individual peptides use a variety of TCR repertoires: Vβ3(4.8%), Vβ5 (19.5%), Vβ8 (38%), Vβ12 (5.4%), and Vβ23 (28.6%) forTARP-27-35-specific CD8⁺ T cells; Vβ3 (13.1%), Vβ5 (12.9%), Vβ8 (19.2%),Vβ12 (3.4%), and Vβ23 (23.6%) for TARP29-37-specific CD8⁺ T cells; Vβ3(7.7%), Vβ5 (7.4%), Vβ8 (16.7%), Vβ12 (16.8%), and Vβ23 (19.3%) forTARP29-37-3A-specific CD8⁺ T cells; and Vβ3 (3.4%), Vβ5 (26.7%) Vβ8(30%), Vβ12 (2.4%), and Vβ23 (23%) for TARP29-37-9V-specific CD8⁺ Tcells. In a phenotype analysis, about 40-60% and 19-40% ofpeptide-specific CD8⁺ T cells in the patients expressed CD45RA andCD45RO, respectively. However, <2% of CD8⁺ T cells express CCR7, and themajority of cells were CD62L^(low), suggesting that most of thepeptide-specific CD8⁺ T cells are activated forms and that the majorityof the memory cells are not central memory CD8⁺ T cells.

Thus, the peptides disclosed herein, specifically TARP-27-35 andTARP-29-37-9V, could be used as a peptide vaccine in adjuvant or aspeptide-pulsed DC in vaccine therapy for prostate and breast cancerpatients who are positive for the HLA-A2 allele. Given that HLA-A2 ispresent in nearly half of the population of North America as well asmuch of the world, and that the expression of TARP is common in prostateand breast cancers, a vaccine containing or expressing these peptidescan be effective in a sizable fraction of prostate and breast cancerpatients. Such vaccine can also be used in combination with otherantigens for prostate or breast cancer, such as PSA and PSMA, to enhancethe efficacy of vaccine therapy. In addition, to enhance CD8⁺ Tcell-mediated immune responses, recombinant vectors including adenovirusor vaccinia virus expressing those antigens can also be used.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. An isolated polypeptide consisting of SEQ ID NO:
 6. 2. An immunogencomprising the polypeptide of claim 1, wherein the polypeptide iscovalently linked to a carrier.
 3. An isolated polynucleotide comprisinga nucleic acid sequence encoding the polypeptide of claim
 1. 4. Thepolynucleotide of claim 3, operably linked to a promoter.
 5. An isolatedvector comprising the polynucleotide of claim
 4. 6. The isolated vectorof claim 5, wherein the vector is a viral vector.
 7. The isolated vectorof claim 6, wherein the vector is a plasmid vector.
 8. An isolated hostcell transformed with the vector of claim
 5. 9. A composition comprisingan immunogenic amount of the polypeptide of claim 1 in a carrier.
 10. Acomposition comprising an immunogenic amount of the polynucleotide ofclaim 3 in a carrier.
 11. A reagent comprising a tetrameric MHC classI/peptide complex comprising the polypeptide of claim 1, wherein thereagent is labeled or unlabeled.
 12. The reagent of claim 11, whereinthe reagent is labeled.
 13. The reagent of claim 11, wherein the labelis a fluorochrome.
 14. A method of detecting CD8 expressing T cells thatspecifically recognize a polypeptide comprising SEQ ID NO: 1 in a sampleisolated from a subject, comprising contacting a sample containingperipheral blood mononuclear cells isolated from the subject with thereagent of claim 11; and detecting the presence of the reagent bound tothe peripheral blood mononuclear cells, thereby detecting CD8 expressingT cells that specifically bind a polypeptide comprising SEQ ID NO: 1 inthe sample.
 15. The method of claim 14, wherein the subject has breastcancer.
 16. The method of claim 14, wherein the subject has prostatecancer.
 17. The method of claim 14, further comprising quantitating thenumber of CD8+ T cells that bind the reagent.
 18. An isolated fusionpolypeptide comprising the amino acid sequence of SEQ ID NO: 6 adjoinedto the amino acid sequence of a heterologous polypeptide.
 19. Anisolated polynucleotide comprising a nucleic acid sequence encoding thepolypeptide of claim
 18. 20. The isolated polynucleotide of claim 19,operably linked to a promoter.
 21. An isolated vector comprising thepolynucleotide of claim
 20. 22. The isolated vector of claim 21, whereinthe vector is a viral vector.
 23. The isolated vector of claim 21,wherein the vector is a plasmid vector.
 24. An isolated host celltransformed with the vector of claim
 21. 25. A composition comprising animmunogenic amount of the polypeptide of claim 18 in a carrier.